Modified Pathogens For Use As Vaccines

ABSTRACT

Described herein are microorganisms that are modified so that they have an increased ability to be recognized by the innate immune system of a eukaryote, relative to an unmodified microorganism. A microorganism may be a gram-negative bacterium that has been modified to produce high potency lipopolysaccharide, e.g.,  Yersinia pestis  expressing LpxL. Such modified microorganisms may be used as vaccines for protection against an infection by the unmodified microorganism. They may also be used as delivery vehicles of one or more heterologous antigens, e.g., antigens from pathogens or those associated with a hyperproliferative eukaryotic cell.

GOVERNMENT SUPPORT

This invention was made with government support under Grant number R01AI057588 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND

The spread of anthrax spores via the mail system in the fall of 2001,followed by the death of several American citizens, demonstrated thatbioterrorism is a highly relevant threat that can have great impact onpeople's health and sense of fear. Plague is among the most contagiousand lethal bacterial diseases with potential for illegitimate use.Historically, plague has been implicated in large epidemics that wereable to change entire societies. Today, smaller plague outbreaks are notuncommon in many countries, and the disease can still induce publicpanic. The latter is exemplified by the suspected outbreak of pneumonicplague in Surat, India in 1994, leading 600 000 people to flee the town(Perry et al., (1997) Clin Microbiol Rev 10:35).

The causative agent of plague was identified in 1894 as theGram-negative bacteria Bacterium pestis, later called Yersinia pestis inhonor of Alexandre Yersin (Perry et al., (1997) Clin Microbiol Rev10:35). Among the species of the genus Yersinia, Y. pestis is mostclosely related to Y. pseudotuberculosis (Achtman et al., (1999) ProcNatl Acad Sci USA 96:14043). Y. pestis is one of history's greatestinfectious killers, with the historic plague epidemics claiming as manyas 200 million lives by some estimates (Perry et al., (1997) ClinMicrobiol Rev 10:35). In the modern US, plague is endemic mainly in thesouthwestern states, with the most cases occurring in New Mexico.Worldwide, a few thousand cases of plague are reported every year, witha mortality rate usually at 5-10% (WHO (1999) Weekly EpidemiologicalRecord 74:340).

Plague is a zoonotic disease; the bacteria spreads naturally viainfected fleas in a rodent reservoir population. The reasons for thecyclic nature of plague epidemics are not defined. However, cycleswithin rodent populations are believed to play an important role. Oncethe flea has transferred the bacteria to its host, Y. pestis spreads toadjacent lymph nodes, where it rapidly multiplies, causing swollen andnecrotic buboes to appear (Butler et al. (1995) In Principles andpractice of Infectious Diseases p. 2070) The bacteria can also spread tothe blood stream, causing septicemia, and in some cases, secondarypneumonic plague. Pneumonic plague can spread directly from person toperson, is highly contagious, and almost 100% lethal when untreated(Ratsitorahina et al. (2000) Lancet 355:111). This is in contrast tobubonic plague, which has a lethality rate of 50-60%. The time fromtransmission to disease onset varies with the route of infection and theindividual, but in general it is assumed an incubation period of 2-6days for pneumonic plague and 1-7 days for bubonic plague.

Outbreaks of pneumonic plague are rare; the last known case where plaguewas spread from person to person in the US was 1924-25 (Perry et al.,(1997) Clin Microbiol Rev 10:35). However, this might be a clinicalcourse of plague were it to occur during a biological attack (Inglesbyet al., (2000) Jama 283:2281). The use of an aerosolized bacteria willcause pneumonic infection. This estimated that 50 kg of Y. pestis spreadwith the wind towards a city of population 5 000 000, will cause 36 000primary deaths and more than 100 000 deaths in total (WHO (1970) HealthAspects of Chemical and Biological Weapons, Geneva, p. 98). In addition,many people would attempt to flee the city, resulting in further spreadof the disease. The establishment of the infection in the rodentpopulation would lead to many subsequent cycles of disease in the area.The mentioned estimate of mortality is based upon treatment of thedisease with appropriate antibiotics. Recent studies have reported theemergence of naturally occurring Y. pestis resistant to multipleantibiotics in Madagascar (Galimand et al., (1997) N Engl J Med 337:677;Guiyoule et al., (2001) Emerg Infect Dis 7:43). The natural spread ofsuch strains, or the potential use of engineered antibiotic resistantstrains in a attack, open the possibility of far more casualities.Therefore, it is of crucial importance to learn more about of the basicbiology of Y. pestis, and how it interacts with the mammalian immunesystem. Through increased knowledge, new therapies against the diseasecan be developed.

Y. pestis harbors a very effective type III secretion system, calledYsc. This system allows attachment of the bacteria to the mammalian cellto occur, with the introduction of a channel from the bacterial and hostcell cytoplasm. This allows translocation of protein effectors from thebacteria to the host cell cytosol. Many of these effectors act tosuppress host cell signaling and phagocytosis (Cornelis et al., (2002) JCell Biol 158:401; Cornelis et al., (2000) Proc Natl Acad Sci USA97:8778). Most of the effector are called Yops, and are encoded by the70 Id) plasmid called pCD1 in the two strains for which the genomicsequence is known, KIM and CO92. For example, YopP/YopJ has the abilityto strongly affect the signaling via the NF-kB and the MAP kinasepathways, both major inducers of inflammatory signals (Cornelis et al.,(2002) J Cell Biol 158:401; Cornelis et al., (2000) Proc Natl Acad SciUSA 97:8778; Cornelis et al., (2000) Proc Natl Acad Sci USA 97:8778).YopH is a powerful phosphotyrosine phosphatase, and can also inhibitphagocytosis. Another potent member of this family is YopE, which hasthe ability to block signaling molecules via GTPase activating protein,affecting Rac, Rho and CdC42.

In spite of the designation of Y. pestis as a NIH/NIAID priority Apathogen, there are currently no licensed US vaccines against plague. Aprevious formalin-killed whole cell vaccine has been discontinued, andsubunit vaccines containing V antigen and F1 capsule protein are stillof an exploratory nature (Titball et al., (2004) Opin Biol Ther 4,965-73). A live EV76-strain vaccine, which is avirulent due to achromosomal deletion in the pgm locus (delta pgm), is still in use inthe former Soviet Union. However, this vaccine may have side effects,and some vaccinated mice have died following inoculation (Titball etal., (2004) Opin Biol Ther 4, 965-73; Russell et al. (1995) Vaccine 13,1551-6). One common problem with plague vaccines, in particular killedvaccines, has been the failure of these to induce protection againstpneumonic plague, the disease form which would be expected following abioterror attack (Titball et al., (2004) Opin Biol Ther 4, 965-73;Russell et al. (1995) Vaccine 13, 1551-6). Thus, there is a need forbetter vaccines to protect against both pneumonic and bubonic plague.

SUMMARY

Provided herein are modified microorganisms, e.g., genetically modifiedmicroorganisms, for use, e.g., as vaccines. The microorganisms may bemodified such that they have an increased ability to be recognized bythe innate immune system of a eukaryote, relative to the microorganismthat is not modified. For example, a modified microorganism may be onethat stimulates a eukaryotic cell receptor involved in innate immuneresponses, e.g., a Toll-like receptor (TLR). Exemplary modificationsinclude the presence of an immunomodulatory molecule, such as a moleculehaving adjuvant activity, e.g., particular lipopolysaccharides (LPS orendotoxin). In one embodiment, Yersinia pestis, the causative agent ofplague, is genetically modified so that it expresses a form of LPS thatstimulates the innate immune system of a eukaryote to stronger levelsthan non modified Yersinia pestis.

Exemplary microorganisms provided herein include gram-negative bacteriathat make low potency lipopolysaccharide (LPS) comprising one or moreheterologous nucleic acids encoding one or more enzymes that permits thebacteria to produce high potency LPS. The high potency LPS preferablyactivates Toll-like receptor 4 (TLR4) in mammalian cells. The highpotency LPS may be hexa-acylated LPS. The heterologous nucleic acid mayencode the LPS biosynthetic enzyme LpxL or a functional analog thereof.The gram negative bacterium may be selected from the group consisting ofYersinia pestis, Chlamydia trachomatis, Francisella tularensis,Legionella pneumophila, Brucella abortus and Chlamydia pneumoniae. Inone example, The gram negative bacterium is Yersinia pestis, and theheterologous nucleic acid encodes the LPS biosynthetic enzyme LpxL or afunctional analog thereof. The enzyme LpxL may be from E. coli and maycomprise, e.g., SEQ ID NO: 4. A gram negative bacterium as describedherein may be alive or killed.

Also provided herein are compositions, e.g., pharmaceutical compositionsand vaccines, comprising at least about 100 or 1,000 colony formingunits (c.f.u.) of a gram-negative bacterium as described herein. Furtherprovided are methods for making a vaccine for protection against agram-negative pathogen, comprising combining a modified gram-negativebacterium and a pharmaceutically acceptable carrier.

Other methods provided herein include methods for protecting a mammalfrom infection by a gram-negative pathogen, comprising, e.g.,administering to a mammal in need thereof an effective amount of avaccine comprising a modified gram-negative bacterium. An exemplarymethod is a method for vaccination of a mammal against the plague,comprising administering to the mammal a therapeutically effectiveamount of a modified Yersinia pestis bacterium.

Also provided herein are methods for delivering an antigen to a subject,e.g., comprising delivering to a subject in need thereof atherapeutically effective amount of a microorganism modified to expressan immunostimulatory molecule, wherein the microorganism furthercomprises an antigen. The antigen may be from a pathogen, or a cell ofthe subject against which an immune response is desired.

The modified microorganisms described herein may also be used in methodsfor identifying an antigen, e.g., in a microorganism. A method maycomprise (i) providing a microorganism modified to express animmunostimulatory molecule; (ii) administering the microorganism to ananimal; (iii) determining the antigenic specificity of an antibody, Bcell and/or T cell of the animal, to thereby identify an antigen in themicroorganism. Step (iii) may further comprise obtaining a sample ofserum from the animal and determining whether one or more antibodies inthe serum are specific for an antigen of the microorganism. A method fordetermining whether a protein is an antigen may also comprise one ormore of the following steps, not necessarily in the order provided: (i)providing a microorganism modified to express an immunostimulatorymolecule and further modified to express one or more proteins; (ii)administering the microorganism to an animal; (iii) determining whetherone or more antibodies, B cells or T cells of the animal are specificfor the one or more proteins. One or more of the proteins may be cancerantigens, antigens associated with an autoimmune disease, and/orantigens from a pathogen.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Y. pestis 37° C. LPS contains inhibitory activity. A) Human PBMCwere treated with 10 or 100 ng/mL of Y. pestis 37° C. LPS or synthetictetra-acyl lipid IV_(A), followed by the addition of Y. pestis 26° C.LPS or E. coli LPS (10 ng/ml) and incubated for 16 hrs. B) HEK 293 cellsexpressing human TLR4/MD2 were exposed to Y. pestis 37° C. LPS (100 and10 ng/ml), followed by addition of 26° C. Y. pestis LPS (10 ng/ml).Cells were incubated for 16 hrs. To study influence on transcriptionfactor activation, HEK293 huTLR4/MD-2 cells were transiently transfectedwith C) NF-κB- or D) IRF-3 dependent 561 promoter-luciferase reporters,treated with Y. pestis 37° C. and/or 26° C. LPS, and incubated for 18hrs. Lipid concentrations are given in ng/ml.

FIG. 2: Structures of lipid A from Y. pestis KIM5 and KIM5(pMW::lpxL).Lipid A was isolated from bacteria grown in liquid culture at 37° C. or26° C. The major forms of lipid A were determined by MALDI-TOF massspectrometry (FIG. 7). Shown are significant lipid A structures found inY. pestis KIM5 at 37° C. A) or 26° C. B), or in KIM5(pMW::lpxL) at bothtemperatures C). Note that a mixture of different compounds may bepresent at a given temperature (FIG. 7).

FIG. 3: Y. pestis KIM5(pMW::lpxL) synthesizes a potent LPS. HEK293 cellsexpressing A) human TLR4/MD2 or B) human TLR2 were stimulated with LPSisolated from Y. pestis KIM5 grown at 26° C. (♦) or 37° C. (▪), Y.pestis KIM5(pMW::lpxL) grown at 26° C. (⋄) or 37° C. (□), or syntheticlipid IV_(A) () for 18 hrs. Supernatants were analyzed by IL-8 ELISA.Human PBMC isolated from a healthy donor were exposed to LPS asdescribed above for 16 hrs before supernatants were examined for C) TNFor D) IL-6 and IL-8 by ELISA. Peritoneal macrophages were isolated fromE) wild type and F) TLR4−/− mice, and stimulated for 16 hrs withdifferent forms of Y. pestis LPS and lipid IV_(A) as described above.

FIG. 4: Y. pestis KIM1001(pMW::lpxL) is avirulent in wild type mice. A)Wild type C57B1/6 mice (n=10 per infection group) were infectedsubcutaneously (s.c.) with either wild type (wt) Y. pestis KIM1001 at1000 c.f.u. (♦) or 100 c.f.u. (▪), or with Y. pestis KIM1001(pMW::lpxL)at 1000 cfu (⋄) or 100 cfu (□). Survival was monitored every 12 hrs.(p<0.001 in comparisons between 1000 c.f.u. LpxL and wt bacteria). B)Wild type mice were infected with increasing doses of KIM1001 andKIM1001(pMW::lpxL). Cumulative results from two* or three** experimentsare shown. C) Wild type C57B1/6 and TLR4−/− mice (n=10 per infectiongroup) were infected s.c. with Y. pestis KIM1001 (▴, ▪) orKIM1001(pMW::lpxL) (□, ⋄) at a dose of 1000 c.f.u. (p<0.001 forKIM1001(pMW::lpxL) vs each of the other groups). D) Colony forming units(c.f.u.) were determined in spleen homogenates from wild type miceinfected intravenously (i.v.) or s.c. with KIM1001 orKIM1001(pMW::lpxL). E) TNF concentration in spleen homogenates from i.v.infected mice from D) were measured by ELISA (p=0.008 between groups).F) Liver sections from wild type or TLR4−/− mice infected s.c. or i.v.with 1000 c.f.u. of KIM1001 or KIM1001(pMW::lpxL) were H&E stained.Arrows indicate bacteria-containing lesions, and the star indicates amicroabcess containing infiltrating inflammatory cells (100×, inserts:1000× magnification). G) Wild type mice were challenged s.c. with 1000c.f.u. of KIM1001(pMW::lpxL) or left untreated. Thirty-five days later,mice were infected s.c. with 1000 c.f.u. of KIM1001 and survival wasmonitored.

FIG. 5. Y. pestis grown at 37° C. is a poor stimulator of TLR4signaling. A) HEK 293 cells stably expressing TLR4/MD-2 or empty vector(pcDNA3) were exposed for 16 hrs to medium alone (white bars) or withheat killed Y. pestis KIM5 grown at 37° C. (host temperature, grey bars)or 26° C. (vector temperature, black bars) at a density of 10⁷ bacteriaper ml. Supernatants were analyzed for IL-8 contents. B) HEK 293TLR4/MD-2 cells were transiently transfected with NF-kB- or IRF-3dependent 561-luciferase reporter constructs and then stimulated withheat killed Y. pestis grown at 37° C. or 26° C. Results are given asfold reporter induction above cells exposed to medium alone.

FIG. 6. Y. pestis 37° C. LPS inhibits activation of non-human primatecells by 26° C. LPS.

Y. pestis KIM5 37° C. LPS or synthetic tetra-acylated lipid IVa wasadded to PBMCs from cynomolgus macaque, followed by addition of KIM5 26°C. LPS. Cells were incubated for 18 hrs and supernatants were analyzedby human IL-8 ELISA. Results indicate that tetra-acylated lipid A/LPSspecies do not activate non-human primate cells, and can inhibitnon-human primate cellular activation by more potent lipid A species ina similar fashion as human cells. Infection of cynomolgus macaque withY. pestis mimics human disease, thus making it a useful non-humanprimate model system for plague pathogenesis. The LD50 for this primateis approximately 400 μm. by inhalation.

FIG. 7 Mass spectrometry analysis of lipid A from Y. pestis KIM5 andKIM5 (pMW::IpxL) grown at 26° C. or 37° C. Wild type Y. pestis KIM5 wasgrown at 26° C. and 37° C. and the lipid A was purified from the wholebacteria by Bligh-Dyer two-phase organic extraction. The lipid was thenpurified over a DE52 column and analyzed by MALDI-TOF mass spectrometry.The negative ion spectra shown here are representative of multipleextractions. Analysis of the positive ion spectra (not shown) was alsodone to determine the location of the arabinose at the reducing end ofthe molecule. Structures are proposed based upon our data compared toprevious reports (12, 13). (A) At 26° C., the bacteria express at leastthree different species of lipid A: tetra-acyl lipid A with four C14:0acyl groups (m/z 1405), a penta-acyl lipid A that has an additionalsecondary C12:0 acyl chain (m/z 1587), and a hexa-acyl lipid A withadditional C12:0 and C16:1 secondary acyl chains (m/z 1824). Anadditional species (Ink 1179) corresponds to a tri-acyl lipid A that mayalso be a fragment ion resulting from the loss of an acyl-linked C14:0group from the tetra-acyl lipid A. In addition, peaks for all four ofthese species with the addition of an 4-amino-4-deoxy-L-arabinose(L-Ara4N) moiety are also observed (m/z 1310, 1537, 1720 and 1955). (B)When the bacteria is grown at 37° C., they produce predominantlytetra-acyl lipid A. Structural analysis was also carried out for thelipid A from Y. pestis KIM5 (pMW::IpxL) expressing the acyltransferaseLpxL from E. coli. (C) At 26° C. the lipid A revealed the presence of asmall amount of tetra-acyl (m/z 1405) and a novel hybrid hexa-acylspecies (ink 1769), which has a C12:0 acyl chain at the 2′ secondaryposition rather than a C16:1 acyl chain. The addition of L-Ara4N wasalso observed (m/z 1900). The peaks at m/z 1349, 1363 and 1377 (observedmore weakly in the other spectra) may not represent tetra-acyl lipid Aheterogeneity, since the mature hexa-acyl species appear as singlepeaks. Hence, they may not be related to lipid A species, and may beother lipids (eg., cardiolipins) recovered from the extraction. (D) At37° C. the mass spectra were similar, predominantly the hexa-acylspecies (m/z 1769) and some penta-acyl lipid A (m/z 1587).

FIG. 8. Non-human primate cells respond to LPS from Y. pestis KIM5(pMW::lpxL) grown at both 26° C. and 37° C. PBMC's isolated fromcynomolgus macaque were stimulated with increasing doses of lipid IV_(A)or LPS isolated from Y. pestis KIM5 or KIM5 (pMW::lpxL) grown at 26° C.and 37° C. for 18 hrs, and supernatant was analyzed by human IL-8 ELISA.The cells did not respond to KIM5 37° C. LPS, but strongly to the otherforms of LPS, in a similar fashion as the response observed in humanPBMC (FIG. 3 c, d).

FIG. 9. The presence of pBR322 does not affect KIM1001 virulence. Wildtype C57BI/6 mice (n=5 per group) were infected with 1000 c.f.u. ofKIM1001 or KIM1001 (pBR322Δtet) s.c. in the nape of the neck Bacteriawere resuspended in 0.05 ml PBS. Survival was monitored every 12 hours.

FIG. 10. Y. pestis KIM1001(pMW::lpxL) infection i.v. is associated withincrease survival times. Wild type c57B1/6 or TLR4−/− mice (n=5 pergroup) were infected with 1000 c.f.u. of KIM 1001 or KIM1001(pMW:lpxL)i.v. in the tail vein. The bacteria were resuspended in a total volumeof 0.5 ml PBS. The animals were monitored every 12 hrs for survival.

FIG. 11. LPS biosynthesis pathway in E. coli (adapted from Raetz et al.(2002) Ann. Rev. Biochem. 71:635).

FIG. 12 shows the structures of exemplary high and low potency LPSmolecules.

FIG. 13 shows that s.c. vaccination with 100000 c.f.u. of KIM1001(pMW::lpxL) protects against subsequent intranasal challenge (pneumonicplague model) with 5000 or 50000 c.f.u. of virulent KIM1001.

DETAILED DESCRIPTION Modifications

Microorganisms may be modified to contain or express animmunostimulatory molecule or adjuvant, e.g., a molecule that stimulatesthe immune system and thereby enhances the recognition of themicroorganism by the immune system. A preferred immunostimulatorymolecule is one that stimulates the innate immune system, generallythrough “innate” receptors. Innate receptors are receptors thatrecognize a wide spectrum of conserved pathogenic components. Examplesof innate receptors include the toll-like receptors (TLRs) and theintracellular nucleotide-binding site leucine-rich repeat proteins(NODs). Conserved pathogenic components are also referred to aspathogen-associated molecular patterns (PAMPs) and their receptors,which are innate receptors, are referred to as pattern recognitionreceptors (PRRs).

TLRs are type I transmembrane proteins that are highly conserved fromman to plants and Drosophila. Most mammalian species have between 10 and15 types of TLRs. Eleven TLRs have been identified in humans (TLR1-11).TLRs function as dimers and they may also depend on other co-receptorsfor full ligand sensitivity. For example, TLR-4 associates with MD-2 torecognize LPS, and CD14 and LPS binding protein (LBP) are also involvedin this process. TLR-4, which is a lipid A co-receptor exists as fourisoforms in humans. The nucleotide and amino acid sequences of isoforms1-4 are set forth as GenBank Accession numbers NM_(—)138554 andNP_(—)612564 (isoform 1 or A); NM_(—)138556 and NP_(—)612566 (isoform 2or B); NM_(—)003266 and NP_(—)003257 (isoform 3 or C); and NM_(—)138557and NP_(—)612567 (isoform 4 or D).

TLR-2 is required for responses to gram-positive bacteria, bacteriallipoproteins, and mycobacteria (Yoshimura et al. (1999). J Immunol163:1; Lien et al. (1999) J Biol Chem 274:33419; Takeuchi et al. (1999)Immunity 11:443; Means et al. (1999) J Immunol 163:3920; Underhill(1999) Nature 401:811; Aliprantis et al. (1999) Science 285:736;Brightbill et al. (1999) Science 285:732; Hirschfeld et al. (1999) JImmunol 163:2382.)

In one embodiment, a microorganism is modified to contain a moleculethat stimulates an innate receptor, such as a TLR or an NOD in aeukaryote, e.g., a human. The stimulation is preferably sufficientlystrong that the administration of a modified microorganism to aeukaryote results in an immune response that is stronger than thatresulting from the administration of the unmodified microorganism to theeukaryote. Even more preferably, the stimulation of the innate receptoris such that the administration of the modified microorganism to theeukaryote will result in an immune response or immunoprotective responsethat will protect the eukaryote from a later infection by thenon-modified microorganism. An “immunoprotective response” is an immuneresponse that results in a decrease of symptoms upon infection with amicroorganism, e.g., a pathogen, or results in a delay or prevention ofa disease associated with infection.

Preferred microorganisms to be modified as described herein arepathogenic microorganisms or infectious agents, such as those thatinduce an undesirable condition or disease in a eukaryote, e.g., amammal. A microorganism may be a prokaryotic or a eukaryoticmicroorganism and may be unicellular or multicellular. Prokaryoticmicroorganisms include bacteria, such as gram-positive andgrain-negative bacteria. Exemplary gram-negative bacteria, includemembers of the Enterobacteriaceae, Vibrionaceae, Francisellaceae,Legionallales, Pseudomonadacea or Pasteurellaceae groups, includingSalmonella spp., Shigella spp., Escherichia spp., Yersinia spp., Vibriospp., Mycobacterium spp. Pathogenic eukaryotic microorganisms includeprotozoans and yeast, such as Candida Albicans, Blastomycosisdermatitidis, Blastomycosis brasiliensi, Coccidioides immitis, andCryptococcus neoformans. A microorganism may also be a virus, e.g.,human immunodeficiency virus (HIV)-1. As further discussed below, thetype of modification made to the mircoorganism may depend on the type ofmicroorganism.

A microorganism may be modified by introducing one or moreimmunostimulatory molecules into the microorganism or by introducinginto the microorganism one or more molecules, such as proteins, that areinvolved in the synthesis of an immunostimulatory molecule. In apreferred embodiment, a microorganism is modified by introducing intothe microorganism one or more nucleic acids encoding one or moreimmunostimulatory molecules or proteins that are involved in thesynthesis of one or more immunostimulatory molecules, such that theencoded proteins are expressed in the microorganism. An exemplarynucleic acid is a heterologous nucleic acid, i.e., a nucleic acid thatis not normally present in the microorganism, or at least not in thatform. It will be understood that it is also possible to modify amicroorganism by directly administrating into it one or moreimmunomodulatory molecules or molecules involved in its synthesis.

In one embodiment, a microorganism is modified such that it contains orproduces an LPS molecule, or lipid A thereof, that stimulates the innateimmune system of a eukaryote. Lipopolysaccharide (LPS, endotoxin) is amajor part of the outer membrane of gram-negative bacteria (Raetz et al.(2002) Annu Rev Biochem 71:635) and is one of the archetypical moleculesrecognized as foreign by the immune system, a pathogen-associatedmolecular pattern. LPS is a highly potent activator of the innate immunesystem, usually with an effect on mononuclear phagocytes at pg/mlconcentrations. In many bacteria, LPS consists of lipid A, adi-glucosamine unit covalently modified with fatty acids and phosphategroups; an oligosaccharide core, and a polysaccharide consisting ofrepeated units of saccharides (also called the O-antigen). Mostbiological effects of LPS can be mimicked by the lipid A portion, alsocalled the “endotoxic core.” The lipid A biosynthetic pathway inEscherichia coli is well studied (Raetz et al. (2002) Annu Rev Biochem71:635). A total of nine enzymes are responsible for the assembly of theE. coli KDO₂-lipid A (FIG. 11).

As further described herein, certain LPS molecules or lipid A portionsthereof, are not strong stimulators of the innate immune system. Forexample, tetra-acylated LPS that is produced by Yersinia pestis at 37°C. (see Examples) is essentially inefficient at stimulating the innateimmune system. Other LPS molecules, such as hexa-acylated LPS, e.g.,shown in FIGS. 2B and 2C, are potent stimulators of the innate immunesystem. Thus, microorganisms are preferably modified to contain a “highpotency LPS” or “strongly stimulatory LPS,” i.e., an LPS that stimulatesthe inmate immune system such that a protective immune response isproduced, as opposed to a “low potency LPS” or “weakly stimulatory LPS.”Typically, there are a few features that may impair lipid A activity: 1)a reduced number of acyl chains (less than six); 2) an increased numberof acyl chains (seven); 3) the length of one or more of the acyl chains(short acyl chains, e.g., less than C10, will impair lipid A activity);long acyl chains: longer than, for example, C18); 4) branching of theacyl chains: more branching will impair lipid A activity; and 5) reducedphosphorylation. As an example, monophosphoryl lipid A is much lessactive and is used as an adjvuant, in natural or synthetic form.Structures of exemplary high potency (strong immune activators) and lowpotency (weak immune activators) LPS structures are set forth in FIG. 12(from Erridge et al. (2004) J. Med. Microbiol. 53:735).

Thus, for example, a high potency LPS or lipid A molecule may comprise 5or 6 acyl chains having a length between about C10 and C18 carbons,e.g., between C12 and C16 carbons. These molecules are preferably notsignificantly phosphorylated and do not have a significant amount ofbranching.

In one embodiment, a microorganism is modified so that it contains oneor more proteins involved in the production of a high potency LPS, suchthat it produces high potency LPS. For example, a microorganism may bemodified by having it express one or more enzymes of the LPS syntheticpathway of a gram-negative bacterium. Exemplary enzymes include theenzymes LpxL, LpxM (also termed HtrB and MsbB, respectively), which are“late” acyl transferases in E. coli and several other gram-negativebacteria, and add the final two secondary acyl chains to thetetra-acylated precursor lipid IVa; and LpxP, a third “late” acyltransferase that is also found in E. coli. LpxL, LpxM and LpxP from E.coli acylates the intermediate (KDO)₂-lipid IVA to form(KDO)₂-(lauroyl)-lipid IVA. A list of LpxL, LpxM, LpxP and otheracyltransferases that are involved in LPS synthesis from variousbacteria and the corresponding GenBank Accession numbers is provided inTable 1 below.

TABLE 1 Description of exemplary acyltransferases of gram-negativebacteria GenBank Enzyme GenBank Acc. Acc. amino Conserved (aliases)Bacteria Gene ID nucleotide seq.* acid seq. regions LpxA Escherichiacoli 944849 U00096 NP_414723 11-70; 109-179; K12 3-262 LpxA Salmonella1251746 AE008705 NP_459233 109-179; 11-70; typhimurium LT2 3-262 LpxAPseudomonas 1044942 AE016779 NP_743760 105-175; 8-68; putida KT24401-258 LpxA Yersinia pestis KIM 1148070 AAM83830 NP_670422 114-179;13-70; 3-262 LpxD Escherichia coli 944882 U00096 NP_414721 104-172;222-285; K12 145-245; 1-337; 16-102 LpxD Salmonella 1251744 AE008705NP_459231 104-172; 222-285; typhimurium LT2 145-245; 18-337; 18-102 LpxDPseudomonas 880525 AE004784 NP_252336 112-183; 203-272; aeruginosa PAO15-340; 7-105 LpxD Yersinia pestis KIM 1148072 AAM83830 NP_670424110-180; 222-285; 145-245; 1-337; 4-102 LpxL Escherichia coli 946216U00096 (complement NP_415572; 4-297 (htrB, b1054, K12 (1114885 . . .1115805)) BAA35863; EG10464, BAA35852; lpxL, waaM) AAC74138 LpxLEscherichia coli 1035020 AE016759 NP_753233 25-318  (htrB) CFT073 LpxLPseudoalteromonas 3710222 CR954246 YP_341083; 6-297 (PSHAa2593)haloplanktis (2738042 . . . 2738965) CAI87641 TAC125 LpxL Candidatus3562698 CP000016 YP_277922; 4-298 (BPEN_423) Blochmannia (501317 . . .502256) AAZ41047 pennsylvanicus str. BPEN LpxL Haemophilus 1491035AE017143 NP_873583; 8-301 (HD1106) ducreyi 35000HP (880973 . . . 881911)AAP95972 LpxL Colwellia 3520374 CP000083 YP_270834; 8-301 (CPS_4183)psychrerythraea (4403276 . . . 4404214) AAZ26125 34H LpxL Rickettsiatyphi str. 2959130 AE017197 YP_067645; 1-290 (RT0704) Wilmington(complement AAU04163 (898361 . . . 899233)) LpxL Salmonella 1252673AE008750 AAL20805; 4-297 typhimurium LT2 NP_460126 LpxL Salmonella3333555 AE017220 AAX65803; 1-270 enterica subsp. YP_216089 entericaserovar Choleraesuis str. SC-B67 LpxL Yersinia 2955847 BX936398YP_071003 4-296 pseudotuberculosis IP 32953 LpxM Escherichia coli 945143U00096 NP_416369; 11-305  (msbB, b1855, K12 (complement AAC74925EG10614, mlt, (1937246 . . . 1938217)) waaN) LpxM Haemophilus 1490398AE017143 NP_872980; 13-307  (HD0404) ducreyi 35000HP (320252 . . .321208) AAP95369 LpxM (mxbB) Salmonella 1070298 AE016837 NP_80481111-305  enterica subsp. enterica serovar Typhi Ty2 LpxM (msbB) Shigellaflexneri 876657 AF348706 NP_085407 9-304 LpxM (msbB) Haemophilus 951108U32705 NP_438368 17-309  influenzae Rd KW20 LpxM (msbB) Yersinia pestisKIM 1147194 AAM85807 NP_669556 12-306  LpxP Escherichia coli 946847U00096 NP_416879; 26-318  (ddg, b2378, K12 (2493601 . . . 2494587)AAC75437 EG12901, G7241) LpxP Erwinia carotovora 2884755 BX950851YP_048893; 7-300 (ddg, subsp. atroseptica (853003 . . . 853935) CAG73695ECA0781) SCRI1043 LpxP Salmonella 1253923 AE008808 NP_461342 6-296 (ddg)typhimurium LT2 LpxP Shigella flexneri 2a 1023448 AE005674 NP_70824826-318  (ddg) str. 301 LpxP Yersinia pestis KIM 1145183 AAM83830NP_667579 32-324  (ddg) *The GenBank accession number provides thenucleotide sequence of the genome of the microorganism and the numbersin parentheses represent the location of the sequence encoding theparticular protein. These numbers are found in the GenBank entry for theprotein.

Whether LpxA, LpxD, LpxL, LpxM and/or LpxP (and/or other enzymesinvolved in LPS synthesis) are introduced into a microorganism willdepend on the microorganism and in particular on whether themicroorganism already contains one or more of these or equivalentenzymes. For example, Yersinia pestis may be modified by administrationof LpxL only, as it already contains the other enzymes that arenecessary for producing high potency LPS. Yersinia pestis strains thatmay be used include the virulent strain KIM1001 or the avirulent strainsKIM5 and EV76, which are avirulent due to the pgm locus deletion. Otherspecies of Yersinia, e.g., Y. pseudotuberculosis, Y. ruckeri, and Y.enterocolitica may also be modified in the same manner as Y. pestis.LpxL may be from E. coli or other bacterium comprising LpxL or afunctional analog or homolog thereof, e.g., those in Table 1.

In one embodiment, a Yersinia strain is modified by introducing into ita nucleic acid encoding E. coli LpxL (SEQ ID NO: 4) or a functionalanalog thereof. For example, a Yersinia strain may be transformed with anucleic acid comprising, consisting essentially of, or consisting of SEQID NO: 1, 2 or 3 or a portion thereof.

Other microorganisms that may be modified to express a high potency LPSare gram-negative bacteria that produce low potency LPS. The followingare examples of such gram-negative bacteria: Francisella tularensis,Chlamydia trachomatis, Legionella pneumophilia, Helicobacter pylori,Bordetella parapertussis, Bordetella pertussis, Brucella abortus,Porphyronzonas gingivalis, Bartonella henselae, Coxiella burnetii,Burkholderia cepecia, Bordetella pertussis, Yersinia pseudotuberculosis,Yersinia enterocolitica, Pseudomonas aeruginosa and Chlamydiapneumoniae. Others are provided elsewhere herein.

TABLE 2 Modifications that can be made to other Gram-negative pathogensfor use as vaccines Enzymes from E. coli or Organism with otherpathogens that can upon expression potentially putative low increaseTLR4 reactivity, decrease virulence TLR4 activation and hence provide astrategy for the generation potential of vaccine strains PseudomonasLpxA, LpxD, LpxM aeruginosa Porphyromonas LpxA, LpxM, LpxD*, LpxK*gingivalis Brucella abortus LpxL, LpxM*, LpxA* Francisella LpxA, LpxM,LpxK tularensis Burkholderia LpxM cepecia Bartonella LpxL, LpxM*henselae Bordetella LpxM, LpxA* pertussis Yersinia LpxL enterocoliticaYersinia LpxL pseudotuberculosis Chlamydia LpxD, LpxL, LpxM, LpxK*trachomatis Coxiella burnetii LpxL, LpxM Bordetella LpxA, LpxMparapertussis Helicobacter LpxA, LpxM, LpxL*, LpxD* pylori BacterioidesLpxD, LpxL, LpxM, LpxK* fragilis Legionella LpxA, LpxD, LpxL, LpxMpneumophila Prevotella LpxM intermedia Burkholderia LpxM cenocepaciaLeptospira LpxA interrogans *next to an enzyme indicates that the enzymemay not be absolutely necessary for modifying the particular pathogen.

Other immunostimulatory molecules of the innate immune system includelipopeptides/lipoproteins, lipoarabinomannan, flagelin from bacterialflagella, double-stranded RNA of viruses, the unmethylated CpG islandsof bacterial and viral DNA, synthetic/modified versions thereof(including oligonucleotides) and certain other single stranded or doublestranded RNA and DNA molecules. Other molecules include bacterial porins(e.g., from Neisseria), lipoteichoic acid, peptidoglycan, syntheticmimics of nucleotides (such as immunostimulants imiquimod andresiquimod/R848 and modified versions thereof). Immunostimulatorymolecules, in particular, synthetic ones may be absorbed onto pathogensin order to increase adjuvant effect. In another example, lipoteichoicacid is introduced into the cell walls of gram-positive bacteria. Theseimmunostimulatory molecules are preferably introduced into the cell wallof a microorganism.

Homologs, portions, and homologs of portions of immunomodulatoryproteins or proteins producing immunomodulatory molecules, such as theproteins listed in Table 1, may also be used. Portions that can be usedinclude biologically active portions, e.g., proteins comprising,consisting essentially of, or consisting of the conserved or activedomains listed in Table 1. Exemplary portions are those comprising,consisting essentially of, or consisting of the catalytically activesite of an enzyme, e.g., the site involved in an acyltransferaseactivity, such as the lipid A acyl transferase domain. Otherbiologically active portions of proteins may be identified by comparisonwith related proteins, which comparison may identify other conserveddomains that are likely to reflect biologically relevant portions of aprotein. Homologs, portions, and homologs of portions of a particularprotein that are biologically active are referred to as “functionalanalogs” or “functional homologs.”

Other homologs include those that differ from a wild-type proteins byone or more amino acids, e.g., about 3, 5, 10, 15, 20 or more aminoacids. In one embodiment, a microorganism is modified to contain aprotein comprising an amino acid sequence that is at least about 80%,85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence (orportion thereof) of a wild-type or naturally occurring immunostimulatoryprotein or protein involved in the synthesis of an immunostimulatoryprotein, such as an enzyme listed in Table 1.

Homologs and analogs can differ from naturally occurring proteins byconservative amino acid differences. For example, conservative aminoacid changes may be made, which although they alter the primary sequenceof a protein or peptide, do not normally alter its function.Conservative amino acid substitutions typically include substitutionswithin the following groups: glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine (in positions other than proteolytic enzymerecognition sites); phenylalanine, tyrosine.

Any number of procedures may be used for the generation of an analog,such as a mutant, a derivative or a variant of a protein using, e.g.,recombinant DNA methodology well known in the art such as, for example,that described in Sambrook et al. (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, New York) and Ausubel etal. (1997, Current Protocols in Molecular Biology, Green & Wiley, NewYork).

A protein may also be modified in a way to form a chimeric moleculecomprising the protein of interest fused to another, heterologouspolypeptide. A fusion or chimeric protein may comprise a polypeptide ofinterest fused to a targeting polypeptide or a peptide that allows easyidentification or localization of the polypeptide of interest. Forexample, a protein may be fused to a “Tag sequence encoding a “Tagpeptide,” such as a hexahistidine tag, myc-epitopes (e.g., see Ellisonet al. (1991) J Biol Chem 266:21150-21157) which includes a 10-residuesequence from c-myc, the pFLAG system (International Biotechnologies,Inc.), the pEZZ-protein A system (Pharmacia, N.J.), and a 16 amino acidportion of the Haemophilus influenza hemagglutinin protein. Furthermore,any peptide can be used as a Tag peptide so long as a reagent, e.g., anantibody interacting specifically with the Tag peptide, is available orcan be prepared or identified. The heterologous polypeptide or peptidepreferably does not interfere with the biological function of thepolypeptide.

In one embodiment, a microorganism is genetically modified, e.g.,modified by introducing into the microorganism one or more nucleic acidsthat encode one or more immunomodulatory molecules or proteins involvedin the production of one or more immunomodulatory molecules. Forexample, a microorganism may be modified by introducing into it one ormore (e.g., 2, 3, 4, or 5) nucleic acids encoding one or more (e.g., 2,3, 4 or 5) enzymes involved in the production of high potency LPS orlipid A, such as the enzymes listed in Table 1, or one or morefunctional analogs thereof. In an illustrative embodiment, a nucleicacid comprising, consisting essentially of, or consisting of anucleotide sequence set forth in Table 1 or a portion thereof encoding afunctional analog, is used. A nucleic acid that may be used maycomprise, consist essentially of, or consist of a nucleotide sequencethat is at least about 80%, 85%, 90%, 95%, 98%, or 99% identical to anucleotide sequence encoding an immunomodulatory protein or a proteininvolved in the synthesis of an immunomodulatory molecule, such as thoselisted in Table 1 or a portion thereof. Other nucleic acids that mayused include those that encode a protein that is at least about 80%,85%, 90%, 95%, 98%, or 99% identical to an immunomodulatory protein orprotein involved in the synthesis of an immunomodulatory molecule, suchas the enzymes set forth in Table 1, or a portion thereof. Yet othernucleic acids that may be used are those that hybridize, e.g., underhigh, low, or medium stringency conditions, to a nucleic acid encoding aprotein described herein, e.g., a nucleic acid encoding all or a portionof a sequence encoding a protein set forth in Table 1. For example, amicroorganism may be modified by introducing into the microorganism oneor more nucleic acids that hybridize to one or more nucleic acidsconsisting of a nucleotide sequence listing in Table 1 or a portionthereof.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. Suchan algorithm is incorporated into the ALIGN program (version 2.0) whichis part of the GCG sequence alignment software package. When utilizingthe ALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused. Yet another useful algorithm for identifying regions of localsequence similarity and alignment is the FASTA algorithm as described inPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. Whenusing the FASTA algorithm for comparing nucleotide or amino acidsequences, a PAM120 weight residue table can, for example, be used witha k-tuple value of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

Hybridizations may be conducted under any of the following conditions:high stringency conditions of 0.2 to 1×SSC at 65° C. followed by a washat 0.2×SSC at 65° C.; low stringency conditions of 6×SSC at roomtemperature followed by a wash at 2×SSC at room temperature;hybridization conditions including 3×SSC at 40 or 50° C., followed by awash in 1 or 2×SSC at 20, 30, 40, 50, 60, or 65° C. Hybridizations canbe conducted in the presence of formaldehyde, e.g., 10%, 20%, 30% 40% or50%, which further increases the stringency of hybridization. Theory andpractice of nucleic acid hybridization is described, e.g., in S. Agrawal(ed.) Methods in Molecular Biology, volume 20; and Tijssen (1993)Laboratory Techniques in biochemistry and molecularbiology-hybridization with nucleic acid probes, e.g., part I chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, New York provide a basic guide to nucleicacid hybridization.

Nucleic acids that are introduced into a microorganism may become partof the chromosome of the microorganism or be present extrachromosomally,e.g. on a plasmid. Methods for introducing and expressing nucleic acids,e.g., expression plasmids, in microorganisms are well known. A varietyof promoters can be used to express a nucleic acid comprising orconsisting of a coding sequence. Similarly a variety of plasmids may beused. Exemplary plasmids include pMW and the mini-Tn7 system.

Exemplary promoters include the promoters that are naturally operablylinked to the gene that is being expressed in the microorganism. Forexample, if E. coli LpxL is expressed in a microorganism, e.g., Yersiniapestis, the natural E. coli promoter of LpxL may be used, e.g.,comprising all or a transcriptionally active portion of SEQ ID NO: 1 or2.

Other promoters for use in Yersinia ssp. include promoters from aYersinia virulon gene. A Yersinia virulon gene is a gene on the YersiniapYV plasmid, the expression of which is controlled both by temperatureand by contact with a target cell. See review by Cornelis et al. (1997).Such genes include genes coding for elements of the secretion machinary(the Ysc genes), genes coding for translocators (YopB, YopD, and LcrV),genes coding for the control elements (YopN and LcrG), and genes codingfor effectors (YopE, YopH, YopO/YpkA, YopM and YopP/YopJ).

Vectors may further comprise other sequence elements such as a 3′termination sequence (including a stop codon and a poly A sequence), ora gene conferring a drug resistance which allows the selection oftransformants, e.g., Yersinia transformants, having received the instantvector.

Nucleic acids, e.g., expression vectors, may be introduced into apathogen, e.g., Yersinia, by a number of known methods. Methods oftransformation of microorganisms, e.g., bacteria, includeelectroporation, calcium phosphate mediated transformation, conjugation,or combinations thereof. For example, a vector can be transformed into afirst bacterial strain by a standard electroporation procedure.Subsequently, such a vector can be transferred from the first bacterialstrain into a second bacterial strain, e.g., Yersinia by conjugation, aprocess also called “mobilization.” Yersinia transformants (i.e.,Yersinia having taken up a nucleic acid) may be selected, e.g., withantibiotics. These techniques are well known in the art. See, forexample, Sory et al. (1994).

After modifying a microorganism, it can be tested for determining itsability to stimulate an immune response, e.g., an innate immune responsein a eukaryote. A modification that increases the recognition of themicroorganism by the immune system of a eukaryote will result in themicroorganism having reduced virulence. Virulence may be reduced by atleast about 10, 10², 10³, 10⁴, 10⁵ or more fold relative to theunmodified microorganism. Tests for virulence depend on the type ofmicroorganism and are known in the art. A modified microorganism mayreduce the likelihood of a eukaryote from being affected by thewild-type microorganism by at least 2, 3, 4, 5 or more orders ofmagnitude. For example, a vaccine may protect at least about 50%, 70%,80%, 90%, 95%, 99% or 99.9% of individuals immunized. Tests fordetermining the virulence of a Yersinia strain and its immunoprotectiveeffect are set forth in the Examples.

Vaccines

Provided herein are compositions, e.g., pharmaceutical compositions,immunostimulatory compositions and vaccines, comprising modifiedmicroorganisms, such as those described herein. Since the modifiedmicroorganisms have reduced virulence and preferably are essentially nonvirulent, they can be used as vaccines to protect a subject from aninfection by the unmodified microorganism.

Vaccine or immunogenic compositions may comprise a modifiedmicroorganism and a pharmaceutically acceptable carrier. Thepharmaceutical carrier or excipient in which the vaccine is suspended ordissolved may be any solvent or solid or encapsulating material such asfor a lyophilized form of the vaccine. The carrier is non-toxic to theeukaryote, e.g., vertebrate, and compatible with the microorganism.Suitable pharmaceutical carriers are known in the art and, for example,include liquid carriers, such as normal saline and other non-toxic saltsat or near physiological concentrations, and solid carriers, such astalc or sucrose. Gelatin capsules can serve as carriers for lyophilizedvaccines. A vaccine may also be presented in the form of an aerosol.Suitable pharmaceutical carriers and adjuvants and the preparation ofdosage forms are described in, Remington's Pharmaceutical Sciences, 17thEdition, (Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1985).

The microorganism in a vaccine may be a live microorganism, i.e., thevaccine is a live vaccine. Alternatively, the microorganism in a vaccineis a killed microorganism. Methods for killing microorganisms forvaccine preparations are well know and include, e.g., heat and/orformalin treatment.

A modified microorganism, especially when used as a live vaccine, mayfurther be attenuated, i.e., weakened, e.g., by mutating themicroorganism to alter its growth capabilities. In one embodiment, anattenuated vaccine is not replication competent or lacks essentialproteins. Methods of making and using live attenuated strains ofbacteria that are suitable for vaccines or immunogenic compositions,including instruction on how to make mutations in virulence genes, aretaught in U.S. Pat. Nos. 5,294,441, 5,387,744, 5,389,368, 5,468,485,5,855,879, and 5,855,880.

Methods for attenuating gram-negative bacteria are described, e.g., inU.S. patent publication number 20050136075. For example, Yersinia can beattenuated by altering its ysc gene, e.g., by creating a deletion in thegene, such that there is reduced secretion or synthesis of Yersiniaouter proteins (Yops) (see also U.S. Pat. No. 5,965,381). A Salmonellaspecies, e.g., S. typhi, S. typhimurium, S. minnesota, S. gallinarum andS. pullorum, may be attenuated by altering one or more of the spa, spi,inv, and ssa genes. A Shigella species, e.g., S. dysenteriae, S. boydi,S. flexneri, and S. sonnei, may be attenuated by altering one or, moreof the spa and mxi genes. An Escherichia species, such as anenterotoxigenic strain (ETEC), an enteropathogenic (EPEC) strain, anenterohemorrhagic (EHEC) strain, a venous thromboses-producing (VTEC)strain, and an enteroinvasive (EIEC) strain, may be attenuated byaltering one or more of the ssc, sep and esc genes. A Pseudomonasspecies, e.g., P. aeruginosa and P. fluorescens, may be attenuated byaltering the psc gene. A Bordetella species, e.g., B. avium, B.pertussis, B. bronchiseptica, and B. parapertussis, may be attenuated byaltering the bsc gene. A Chlamydia species, e.g., C. psittaci, C.trachomatis, and C. pneumoniae, may be attenuated by altering one ormore of the bsc and cds genes. A Vibrio species, e.g., V. cholerae, V.cholerae O1, V. cholerae non-O1, V. vulnificus, and V. parahaemolyticus,may be attenuated by altering the bsc gene. Attenuation may also be bymutation to the kasugamycin resistance, e.g., by introducing a gene forkasugamycin resistance (Mecsas et al., 2001, Inf 1 mm 67: 2779-2787).

The vaccine, immunogenic composition may also comprise a“balanced-lethal host-vector system,” which would enables themicroorganism to maintain the presence of the ectopic polynucleotideconstructs without the need for external selection (see U.S. applicationpublication No. 20040101531, U.S. Pat. Nos. 5,294,441, 5,387,744,5,424,065, 5,656,488, 5,672,345, 5,840,483, 5,855,879, 5,855,880 and6,024,961, and PCT/US01/13915). The balanced-lethal host-vector systemis based upon the concept of having an inactivating mutation in anessential gene of the microorganism, wherein a functional copy of theessential gene is provided on a plasmid (a genetically engineeredautonomous extrachromosomal polynucleotide), which contains thepolynucleotide that encodes an immunostimulatory protein or proteininvolved in the synthesis of an immunostimulatory molecule. Thus, inorder for the microorganism to remain viable, the plasmid, whichcontains the functional copy of the essential gene and thepolynucleotide that encodes the protein of interest, must be maintainedin the carrier bacterium. In one embodiment, the essential gene encodesβ-aspartate semialdehyde dehydrogenase (Asd), and the plasmid encodes afunctional Asd polypeptide, which complements the chromosomal asdmutation, but which cannot replace the defective chromosomal Asd gene byrecombination. Lack of a functional Asd polypeptide causes bacterialcells to lyse. The polynucleotide encoding the functional Asdpolypeptide and the polypeptide that encodes the protein of interest arephysically linked on the same episome, thereby ensuring that the carrierbacteria maintains the polynucleotide that encodes the protein ofinterest.

A modified microorganism may also comprise one or more additionalheterologous proteins, such as antigens or other immunomodulatoryproteins, that will further enhance the immunogenicity of the modifiedmicroorganism. For example, a modified microorganism may encode acytokine or lymphokine that could enhance an immune response. Theseadditional modifications may, e.g., stimulate the adaptive immuneresponse, such as cellular and/or humoral immune responses.

Also provided herein are methods for making a vaccine for immunizing asubject population against infection by a pathogen. The method maycomprise providing the pathogen in a modified form as described hereinand a pharmaceutically acceptable carrier. Methods for making a modifiedmicroorganism may comprise introducing into the microorganism a nucleicacid encoding an immunostimulatory protein or a protein involved in thesynthesis of an immunomodulatory molecule.

Also provided are kits, e.g., kits for immunizing a subject againstinfection by a pathogen. A kit may comprise at least one unit dose of amodified pathogen. A unit dose may be an effective dose to provideimmunization against infection by a wild-type strain. Exemplary dosesare about 10², 10³, 10⁴, 10⁵ or at least about 10⁶ colony forming units(c.f.u.). A vaccine may be provided in a delivery vehicle or container,such as a syringe.

Thus, vaccines based on modified microorganisms as described herein maybe used for treating or preventing a condition or disease that ismediated or exacerbated by the corresponding unmodified microorganism.Table 3 set forth below lists various diseases and their causativeagents.

TABLE 3 Exemplary microbial diseases and their gram-negative causativeagent Microorganism Disease Yersinia pestis bubonic and pneumonic plagueYersinia enterocolitica broad range of gastrointestinal syndromesYersinia adenitis and septicaemia pseudotuberculosis Yersinia ruckerienteric redmouth disease (ERM) in salmonids and a few other freshwaterfish Bordetella pertussis whooping cough Bordetella parapertussisrespiratory disease in humans, e.g., whooping cough and mildpharyngitis, and sheep Chlamydia pneumoniae acute respiratory disease,and contributing to atherosclerosis and heart disease Chlamydiatrachomatis conjunctivitis, trachoma (infectious cause of blindness),urethritis, pelvic inflammatory disease (PID) Pseudomonas aeruginosaurinary tract infections, respiratory system infections (in particularin cystic fibrosis patients), dermatitis, soft tissue infections,bacteremia, bone and joint infections, gastrointestinal infections and avariety of systemic infections Francisella tularensis Tularemia,ulceroglandular tularemia, oculoglandular tularemia, pulmonarytularemia, typhoidal tularemia Legionella pneumophilia Legionnaire'sdisease Helicobacter pylori duodenal ulcers, gastric ulcers, stomachcancer, non-ulcer dypepsia Brucella abortus Brucellosis (Malta fever)Porphyromonas gingivalis periodontal disease Bartonella henselae CatScratch Disease and bacillary angiomatosis, bacteremia, and sepsis inimmunocompromised patients Coxiella burnetii Q fever Burkholderiacepecia respiratory system infections, in particular in cystic fibrosispatients

Also provided herein are methods for protecting a subject from apathogen induced condition or disease, e.g., methods of providingimmunity from infection by a wild-type pathogen, e.g., Yersinia pestis,to a subject. A method may comprise administering to a subject, e.g., asubject in need thereof, a therapeutically effective amount of amodified form of the microorganism causes a condition or disease (thepathogen), wherein the dose confers immunity on the subject to a diseasestate caused by the wild-type microorganism. A person in need thereofmay be a person that has been exposed to or is likely to be exposed toor infected with a pathogenic microorganism or infectious agent, such asmedical personel or personal that has been exposed to or likely to beexposed to bioterrorism. Other persons at risk of being exposed include,but are not limited to, military personnel, mail handlers, andgovernmental officials, as well as those with weakened immune systems,for example, the elderly, people on immunosuppressive drugs, subjectswith cancer, and subjects infected with HIV.

A vaccine described herein may also be used for reducing the frequencyof incidence of a disease that it transmitted by a non-human animal,e.g., plague, in a human population that is contiguous to an animalpopulation reservoir. A method may comprise administering to the animalpopulation a modified microorganism described herein. For example, itmay be desirable to immunize rats, which transmit the plague, with amodified Yersinia pestis bacterium.

Vaccines or immunogenic compositions may be administered to eukaryotes,such as vertebrates, e.g., mammals, including humans, canines, felines,ovines, bovines, equines, sheep, cattle, livestock and poultry accordingto several methods.

Methods of administration of a modified microorganism, e.g., in avaccine preparation, include oral administration, gastric intubation orintranasal administration, e.g., in the form of aerosols, intravenous,intramuscular or subcutaneous injection, vaginal or rectaladministration, parenteral administration, for example, intraperitoneal,intraperitoneal, intrasternal, or intra-articular injection or infusion,or by sublingual, oral, topical, or transmucosal administration, bypulmonary inhalation, or through whole body spray (see, e.g., WO00/04920).

The compositions described herein are preferably given to an individualin a “prophylactically effective amount” or a “therapeutically effectiveamount,” this being sufficient to show benefit to the individual. Theactual amount administered, and rate and time-course of administration,will depend, e.g., on the nature and severity of what is being treatedor prevented and on the type of microorganism. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors.

An effective dose of a vaccine may provide immunity to a virulentpathogen by at least about 2, 3, 4, or 5 or more orders of magnitudemore than the level of immunity in a non-immunized subject. An effectivedose may comprise, e.g., about 10³ to about 10¹² microorganisms per kgbody weight of a subject, such as about 10⁴ to about 10⁸ microorganismsper kg body weight of a subject. An effective dose may also comprisesabout 10⁸ to about 10¹² microorganisms per kg body weight of thesubject.

Multiple dosages may be used as needed to provide the desired level ofprotection. For example, one or more boosters may be needed over time tomaintain protection of a eukaryote.

The level of protection provided to a subject after immunization may bedetermined by methods known in the art, such as by determining the levelof antibodies and/or T cells (such as cytotoxic T lymphocytes (CTL))specific for antigens from the microorganisms, produced in response tothe immunization. The presence of specific CTLs can be detected usingstandard assays such as an assay for Cr⁵¹ release or for the secretionof IFN-γ. The presence of specific antibodies can be detected by assayssuch as ELISA using the antigens which are immobolized on a cultureplate, or a standard proliferation assay for T-helper cells.

Adjuvants may be added to enhance the antigenicity of a modifiedmicroorganism if desired, but are generally not required to induce aneffective immune response, since components of the modifiedmicroorganisms generally serve as adjuvants. Other agents that may beadministered to a subject that is being treated with a vaccine describedherein include anti-infectious agents, e.g., anti-fungal compounds,anti-viral compounds, and antibiotics. Antibiotics include, but are notlimited to, amoxicillin, clarithromycin, cefuroxime, cephalexinciprofloxacin, doxycycline, metronidazole, terbinafine, levofloxacin,nitrofurantoin, tetracycline, and azithromycin. Anti-fungal compounds,include, but are not limited to, clotrimazole, butenafine, butoconazole,ciclopirox, clioquinol, clioquinol, clotrimazole, econazole,fluconazole, flucytosine, griseofulvin, haloprogin, itraconazole,ketoconazole, miconazole, naftifine, nystatin, oxiconazole, sulconazole,terbinafine, terconazole, fluconazole, and tolnaftate. Anti-viralcompounds, include, but are not limited to, zidovudine, didanosine,zalcitabine, stavudine, lamivudine, abacavir, tenofovir, nevirapine,delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir,saquinavir, amprenavir, and lopinavir. Anti-infectious agents alsoinclude hyper-immune globulin. Hyper-immune globulin is gamma globulinisolated from a donor, or from a pool of donors, that has been immunizedwith a substance of interest. Specifically, hyper-immune globulin isantibody purified from a donor who was repeatedly vaccinated against apathogen.

When an agent is administered to a eukaryote that is being administereda modified microorganism, administration of the agent and the modifiedmicroorganism may be done simultaneously or sequentially.

Use of Modified Microorganisms as Delivery Vehicles for HeterologousMolecules

The modified microorganisms described herein may also be used asdelivery systems, e.g., vaccine delivery systems. This use is based atleast in part on the fact that the modified microorganisms have beenrendered avirulent and will provide strong adjuvant activity and antigenpresentation. In one embodiment, a microorganism modified to express animmunostimulatory molecule is further modified to comprise or expressone or more macromolecules, e.g., heterologous macromolecules, againstwhich an immune response is desired. A “heterologous molecule” in amicroorganism is a molecule that is not naturally present, e.g., in thesame form or amount, in the microorganism. An exemplary macromolecule isa protein, such as a pathogenic antigen. Thus, for example, Y. pestisexpressing LpxL that also comprises or expresses an antigen from anotherpathogen, e.g., a bacteria, fungus or virus, may be used to vaccinate asubject and thereby protect the subject from the pathogen. Otherproteins that can be delivered to a subject using the modifiedmicroorganisms described herein include cancer antigens and antigensassociated with autoimmune diseases. The macromolecule, e.g., protein,expressed in the modified microorganism may be one that is known in theart or one that has been identified as further described herein.

Exemplary molecules from pathogens that may be delivered using thevaccine system described herein include antigens from the NationalInstitute of Allergy and Infectious Diseases (NIAID) priority pathogens.These include Category A agents, such as variola major (smallpox),Bacillus anthracia (anthrax), Yersinia pestis (plague), Clostridiumbotulinum toxin (botulism), Francisella tularensis (tularaemia),filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagic fever),arenaviruses (Lassa (Lassa fever), Junin (Argentine hemorrhagic fever)and related viruses); Category B agents, such as Coxiella burnetti (Qfever), Brucella species (brucellosis), Burkholderia mallei (glanders),alphaviruses (Venezuelan encephalomyelitis, eastern & western equineencephalomyelitis), ricin toxin from Ricinus communis (castor beans),epsilon toxin of Clostridium perfringens; Staphylococcus enterotoxin B,Salmonella species, Shigella dysenteriae, Escherichia coli strainO157:H7, Vibrio cholerae, Cryptosporidium parvum; and Category C agents,such as nipah virus, hantaviruses, tickborne hemorrhagic fever viruses,tickborne encephalitis viruses, yellow fever, and multidrug-resistanttuberculosis.

Exemplary antigens that may be delivered include the following ones: B.anthracis protective antigen (PA); influenza hemagglutinin (HA), such asfrom H5N1 influenza; and Yersinia pestis LcrV and Caf1. Additionaltoxins produced by NIAID priority pathogens that are potential targetsfor this vaccine strategy include ricin, using as antigen a non-toxicricin mutant selected in yeast (Allen et al. (2005) Yeast 22:1287); aC-terminal heavy chain fragment from botulinum neurotoxin serotype E(Dux et al. (2005) Protein Expr. Purif, in press); and the B subunit ofthe type II shiga toxin that is the dominant hemorrhagic toxin producedby most enterohemorrhagic strains of E. coli (Marcato et al. (2005)Infect. Immun. 73:6523).

Other toxins that may be protected against as described herein, e.g., byincluding in the modified microorganisms inactivated forms of toxins,such as anatoxin antigens, including toxoids (inactivated but antigenictoxins), and toxoid conjugates include: pertussis toxoid,Corynebacterium diphtheriae toxoid, tetanus toxoid, Haemophilusinfluenzae type b-tetanus toxoid conjugate, Clostridium botulinum Dtoxoid, Clostridium botulinum E toxoid, toxoid produced from Toxin A ofClostridium difficile, Vibrio cholerae toxoid, Clostridium perfringensTypes C and D toxoid, Clostridium chauvoei toxoid, Clostridium novyi(Type B) toxoid, Clostridium septicum toxoid, recombinant HIV tat IIIBtoxoid, Staphylococcus toxoid, Actinobacillus pleuropneumoniae Apx Itoxoid, Actinobacillus pleuropneumoniae Apx II toxoid, Actinobacilluspleuropneumoniae Apx III toxoid, Actinobacillus pleuropneumoniae outermembrane protein (OMP) toxoid, Pseudomonas aeruginosa elastase toxoid,snake venom toxoid, Mannheimia haemolytica toxoid, Pasteurella multocidatoxoid, Salmonella typhimurium toxoid, Pasteurella multocida toxoid, andBordetella bronchiseptica toxoid. Recombinant methods of converting atoxin to a toxoid are known in the art (see, e.g., Fromen-Romano, C., etal., Transformation of a non-enzymatic toxin into a toxoid by geneticengineering, Protein Engineering vol. 10 no. 10 pp. 1213-1220, 1997).

Exemplary viruses from which to protect organisms using modifiedmicroorganisms include Coxsackie viruses, cytomegaloviruses,Epstein-Barr viruses, flaviviruses, hepatitis viruses, herpes viruses,influenza viruses, measles viruses, mumps viruses, papilloma viruses,parainfluenza viruses, parvoviruses, rabies viruses, respiratorysyncytial viruses, retroviruses, varicella viruses, adenoviruses, arenaviruses, bunyaviruses, coronaviruses, hepadnaviruses, myxoviruses,oncogenic viruses, orthomyxoviruses, papovaviruses, paramyxoviruses,parvoviruses, picornaviruses, pox viruses, rabies viruses, reoviruses,rhabdoviruses, rubella viruses, togaviruses, equine herpes virus 1,equine arteritis virus, IBR-IBP virus, and BVD-MB virus. Other virusesinclude leukemia, lymphotrophic, sarcoma, lentiviruses and otherimmunodeficiency or tumor viruses. Exemplary lymphotrophic virusesagainst which a subject may be protected include T-lymphotrophicviruses, such as human T-cell lymphotrophic viruses (HTLVs, such asHTLV-I and HTLV-II), bovine leukemia viruses (BLVS) and feline leukemiaviruses (FLVs). Particularly preferred lentiviruses include human (HIV),simian (SIV), feline (FIV) and canine (CIV) immunodeficiency viruses,with HIV-1 and HIV-2 being even more preferred.

Examples of viral antigens to be used include env, gag, rev, tar, tat,nucleocapsid proteins and reverse transcriptase from immunodeficiencyviruses (e.g., HIV, FIV); HBV surface antigen and core antigen; HCVantigens; influenza nucleocapsid proteins; parainfluenza nucleocapsidproteins; human papilloma type 16 E6 and E7 proteins; Epstein-Barr virusLMP-1, LMP-2 and EBNA-2; herpes LAA and glycoprotein D; as well assimilar proteins from other viruses.

Modified microorganisms comprising an antigen may also be used inmethods for conferring a broad based protective immune response againsthyperproliferating cells that are characteristic of hyperproliferativediseases, as well as a method of treating individuals suffering fromhyperproliferative diseases. As used herein, the term“hyperproliferative diseases” is meant to refer to those diseases anddisorders characterized by hyperproliferation of cells. Examples ofhyperproliferative diseases include all forms of cancer and psoriasis.In an illustrative embodiment, a method is for treating a subject, suchas a subject having or likely to develop a hyperproliferative disease,and comprises administering to the subject a therapeutically effectiveamount of a modified microorganism expressing an hyperproliferatingcell-associated protein or functional homolog thereof. Thehyperproliferating cell-associated protein or functional homolog thereofpreferably induces an immune response against a cell comprising thehyperproliferating cell-associated protein. As used herein, the term“hyperproliferative-associated protein” is meant to refer to proteinsthat are associated with a hyperproliferative disease.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it is preferred that the protein isproduced exclusively and/or at higher levels in hyperproliferative cellsrelative to normal cells. A hyperproliferative-associated protein may bethe product of a mutation of a gene that encodes a protein. A mutatedgene may encode a protein that is nearly identical to the normal proteinexcept it has a slightly different amino acid sequence, which results ina different epitope not found on the normal protein. Such targetproteins include those which are proteins encoded by oncogenes such asmyb, myc, fyn, and the translocation genes bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target proteins for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas, andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune diseases.Other tumor-associated proteins can be used as target proteins, such asproteins which are found at higher levels in tumor cells, including theprotein recognized by monoclonal antibody 17-1A and folate bindingproteins.

Exemplary T cell mediated autoimmune diseases include rheumatoidarthritis (RA), multiple sclerosis (MS), Sjogren's syndrome,sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmunethyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma,polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener'sgranulomatosis, Crohn's disease and ulcerative colitis. Each of thesediseases is characterized by T cell receptors that bind to endogenousantigens and initiate the inflammatory cascade associated withautoimmune diseases. Vaccination against the variable region of the Tcells would elicit an immune response including CTLs to eliminate thoseT cells.

In RA, several specific variable regions of T cell receptors (TCRs)which are involved in the disease have been characterized. These TCRsinclude Vβ-3, Vβ-14, Vβ-17 and Vβ-17 (see, e.g., Howell, M. D., et al.,1991 Proc. Natl. Acad. Sci. USA 88:10921-10925; Paliard, X., et al.,1991 Science 253:325-329; Williams, W. V., et al., 1992 J. Clin. Invest.90:326-333). Thus, vaccination with a modified microorganism thatdelivers at least one of these proteins or a functional homolog thereofis expected to elicit an immune response that will target T cellsinvolved in RA.

In MS, several specific variable regions of TCRs which are involved inthe disease have been characterized. These TCRs include Vβ-7 and Vα-10(see, e.g., Wucherpfennig, K. W., et al., 1990 Science 248:1016-1019 andOksenberg, J. R., et al., 1990 Nature 345:344-346). Thus, vaccinationwith a modified microorganism that delivers at least one of theseproteins or a functional homolog thereof is expected to elicit an immuneresponse that will target T cells involved in MS.

In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs includeVβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 andVα-12. Thus, vaccination with a modified microorganism that delivers atleast one of these proteins or a functional homolog thereof is expectedto elicit an immune response that will target T cells involved inscleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Vaccines can be prepared usingthis information.

Exemplary B cell mediated autoimmune diseases against which modifiedmicroorganisms comprising an antigen may protect a subject include Lupus(SLE), Grave's disease, myasthenia gravis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliarysclerosis and pernicious anemia. Each of these diseases is characterizedby antibodies which bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of such antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity may have to be identified. If this is the case, abiopsy can be performed and samples of the antibodies present at a siteof inflammation can be taken. The variable region of those antibodiescan be identified using standard techniques and vaccines can be preparedusing this information.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes the variableregion of such anti-DNA antibodies found in the sera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al. 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md. In addition, a general method for cloning functionalvariable regions from antibodies can be found in Chaudhary, V. K., etal., 1990 Proc. Natl. Acad. Sci. USA 87:1066.

Modified microorganisms that are used as delivery vehicles forheterologous antigens may be prepared and administered as furtherdescribed herein for modified microorganisms.

Methods for Identifying Antigens

Also provided herein are methods for identifying new antigens for use invaccines. A method may comprise administering to an animal a modifiedmicroorganism as described herein and identifying one or more antigensfrom the microorganism to which the animal raised an immune response,e.g., by determining the antigenic specificity of antibodies in theserum of the animal. The methods may be based at least in part on thefollowing theory. There is a definite linkage between stimulation ofinnate immunity and the character and quality of the subsequent adaptiveimmune response. Hence, pathogens which don't stimulate innate immunityvery well may produce an adaptive response skewed to a limitedrepertoire of antigens. A common approach to antigen discovery beginswith the analysis of the antigen specificity of B and T cells fromindividuals who have recovered from infection, on the assumption thatthese individuals will have adaptive responses to appropriate antigens.If the ability of the pathogen to avoid stimulating innate immunityskews antigenic responses, it may hide valuable antigens from discoveryby this approach. Incorporation of adjuvant-like activity into thepathogen, e.g., a bacterium, is expected to enhance the range andstrength of specific responses, revealing antigens that might notproduce significant response with the wild type pathogen. For example,in Y. pestis, the development of subunit vaccines has been limited bythe availability of only two antigens that have been clearly shown to beeffective: LcrV (V-antigen) and Caf1 (F1). The methods described herein,such as comprising administering to an animal an LpxL expressing Y.pestis, may be used to identify other antigens of Y. pestis that may beused in vaccines, e.g., in subunit vaccines.

An exemplary method for identifying an antigen in a microorganismcomprises (i) providing a microorganism modified to express animmunostimulatory molecule; (ii) administering the microorganism to ananimal; (iii) determining the antigenic specificity of an antibody, Bcell and/or T cell of the animal, to thereby identify an antigen in themicroorganism. Determining the antigenic specificity of an antibody, Bcell and/or T cell may comprise first obtaining a sample of serum orblood from the animal. Determining the antigenic specificity may alsocomprise contacting an antibody, B cell or T cell or portion thereofhaving antigen binding capacity, with one or more macromolecules, e.g.,proteins, or portions thereof of the microorganism and determiningwhether the antibody, B cell or T cell or portion thereof interact withthe one or more macromolecules or portions thereof. A method may furthercomprise formulating the macromolecule or portion thereof in a vaccineformulation for administering to a subject in need of protection againstthe microorganism comprising the macromolecule.

In an alternative embodiment, a method comprises using a microorganism(the “first microorganism”) modified to express an immunostimulatorymolecule, wherein the microorganism is further modified to express oneor more macromolecules, e.g., proteins, or fragments thereof of anothermicroorganism (the “second microorganism”). For example, for identifyingantigens of Francisella tularensis, a method may comprise using Y.pestis expressing LpxL and further expressing one or more proteins orportions thereof of Francisella tularensis. The method may furthercomprise administering this doubly modified microorganism to an animaland determining whether the animal produced any antibodies or T or Bcell that is specific for one or more proteins of Francisellatularensis.

In yet another embodiment, a method comprises using a microorganismmodified to express an immunostimulatory molecule, wherein themicroorganism is further modified to express one or more molecules of avirus, one or more cancer antigens, or any molecule that could be apotential antigen recognized by the immune system of an animal. Forexample, a method may comprise (i) providing a microorganism that ismodified to express an immunostimulatory molecule, wherein themicroorganism is further modified to express one or more virus or cancerantigens or portions thereof; (ii) administering the modifiedmicroorganism to an animal; and (iii) determining whether the animalraised any antibodies or T or B cell response against one or more of thecancer antigens. Virus or Cancer antigens against which an immuneresponse (e.g., antibodies, T or B cell response) was raised may furtherbe formulated into a vaccine for administration into a subject fortreating or preventing the cancer that is associated with the particularcancer antigen. Similarly, antigens of any undesirable cells may beidentified by the methods described herein, such as antigens associatedwith autoimmune diseases, which antigens can then be used in thetreatment or prevention of the autoimmune disease.

The microorganisms that may be used in the methods for identifying anantigen include any of the modified microorganisms described herein,such as Y. pestis expressing LpxL, or other microorganisms modifiedfollowing the same principle. Animals to which the microorganisms may beadministered include any animal, e.g., a mammal, that is capable ofraise an immune response against an antigen, such as rodents, e.g., miceand rats, and rabbits. A microorganism may be administered to the animalin any manner that is likely to result in the animal raising an immuneresponse against the microorganism, e.g., by oral or nasaladministration or by parenteral administration, such as intramuscular,subcutaneous or intraperitoneal injection. After a period of timesufficient for the animal to have responded immunologically to thepresence of the microorganism, e.g., at least about 7 days, 10 days, 14days, 21 days or one month, a serum, cell or blood sample may becollected according to methods known in the art. Since the amount andaffinity of antibodies (e.g., IgG and IgM) may increase with repeatedimmunizations, one can also immunize an animal at least 2, 3 or 5 times,prior to obtaining a sample to determine antigenic specificity.

Determining the antigenic specificity of antibodies, e.g., present inthe serum or blood of an animal, may be conducted according to methodswell known in the art. A method may comprise contacting an antibody orblood or serum or cell sample of an animal with a candidatemacromolecule, such as a naturally occurring protein of themicroorganism or a portion thereof (e.g., a fragment of at least about 6amino acids, 10 amino acids, 30 amino acids, 50 amino acids or more)under conditions in which an antibody would bind to an antigenrecognized by the antibody. The candidate macromolecule may be labeled,such as with a fluorochrome. A method may further comprise detecting thebinding of a candidate macromolecule to the antibody. Similar methodsmay be used for determining the antigen-binding specificity of a B or Tcell. Furthermore, reactive cell subsets may be used in proliferationassays following exposure to the specific antigen.

Antigens identified as described herein may be prepared in vaccineformulations. For example, one or more antigens or portion thereof, maybe combined with a pharmaceutically acceptable carrier. Alternatively,one or more nucleic acids encoding one or more antigens or portionthereof may be formulated into a therapeutic composition andadministered to a subject in need thereof. A nucleic acid may comprise apromoter that is operably linked to the sequence that encodes an antigenor portion thereof. A nucleic acid may be in the form of a vector, e.g.,an expression vector.

It is possible to express at least about 2, 3, 5, 10, or 25 proteins ina microorganism, thereby increasing the likelihood of identifying one ormore of these proteins as antigens that can be used in vaccines. Asanother way to adapt this method to a high throughput method, librariesof macromolecules may be tested or screened for binding of antibodies, Bcells or T cells of the animal to whom a modified microorganism wasadministered.

The present description is further illustrated by the followingexamples, which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications and GenBank Accessionnumbers as cited throughout this application) are hereby expresslyincorporated by reference. When definitions of terms in documents thatare incorporated by reference herein conflict with those used herein,the definitions used herein govern.

EXAMPLES Example 1 Evasion of Endotoxin Signaling is Critical for theVirulence of Yersinia pestis Summary

The Gram-negative bacterium Yersinia pestis, causative agent of plague,efficiently escapes containment by the innate immune system. Toll-likereceptor-4 (TLR4) is central in innate recognition of gram-negativeendotoxin/lipopolysaccharide (LPS). Y. pestis LPS is highly inflammatoryin bacteria grown at temperatures prevailing in its flea vector, butweakly stimulatory during growth at 37° C. Here we describe how theresulting evasion of TLR4-signaling is required for Y. pestis virulence.When modified to produce potent LPS at 37° C., normally lethal 1′.pestis is rendered avirulent for wild type mice, but remains virulent inTLR4−/− animals. The modified strain also acts as a vaccine againstvirulent Y. pestis.

One-Sentence Summary

The Gram-negative pathogen Yersinia pestis, the causative agent ofplague, modifies its lipopolysaccharide at host temperature (37° C.),and we find that this modification is necessary for its virulence byevasion of Toll-like receptor 4 signaling.

Body Text:

The Gram-negative bacterium Yersinia pestis, causative agent of plague,produces a fulminant systemic infection following inoculation of a fewbacteria into the skin by flea bite (1). The key to the extremevirulence of Y. pestis is escape from innate antibacterial defenses (1,2). The activation of Toll-like receptor 4 (TLR4) by endotoxin(lipopolysaccharide, LPS), a major component of the Gram-negative outermembrane, is a major pathway inducing host innate immune responses tonative bacteria (3-7). The immune-activating moiety of LPS is lipid A, adi-glucosamine unit with covalently attached acyl chains, whichinteracts with TLR4 and MD-2 to induce cellular responses (3-8). Lipid

A structure is conserved among Gram-negatives but is not invariant. Thenumber and structure of acyl chains varies among species, is influencedby environment, and is often heterogeneous even within a single species(6, 9, 10). It has been hypothesized that production of weaklystimulatory LPS plays a role in virulence of several Gram-negativepathogens (10). Hexa-acylated structures are found in Y. pestis grown at21° C. to 27° C. (ambient/flea temperatures), but LPS is predominantlytetra-acylated at 37° C. (host temperature) (11-13). Hexa-acylatedLPS/lipid A is normally a strong activator of human cells, whereastetra-acylated lipids have lower stimulatory activity (4, 11, 12, 14,15). It has been suggested that the temperature-dependent remodeling oflipid A structure could be necessary for Y. pestis to achieve the highbacterial load in mammalian blood required for efficient flea infectionprior to the induction of lethal shock. Y. pestis may also need tominimize TLR4 stimulation early in infection to prevent containment bylocal inflammation.

Y. pestis grown at 37° C. poorly activated TLR4 signaling (FIG. 5).Since tetra-acylated LPS may antagonize activity induced by potentendotoxin (4, 14, 15), we also analyzed the anti-inflammatory role of37° C. Y. pestis LPS. LPS purified from Y. pestis KIM5 grown at 26° C.,but not 37° C. (16), activated human peripheral blood mononuclear cells(PBMC) (FIG. 1A) and HEK293 huTLR4/MD-2 cells (FIG. 1B). Activationinduced by 26° C. Y. pestis LPS and Escherichia coli LPS was inhibitedby 37° C. Y. pestis LPS and by the synthetic tetra-acylated lipid IV_(A)in human PBMC (FIG. 1A). Stimulation of HEK293 huTLR4/MD-2 cells assayedby interleukin (IL)-8 release (FIG. 1B) and activation of NF-κB andIRF-3 dependent reporters (FIG. 1C,D) by the 26° C. LPS was alsoinhibited by the 37° C. preparation. We also observed repression ofcytokine release in PBMC from cynomolgus macaque (Macaca fascicularis)(FIG. 6), a model organism for studies of plague in primates. Bacteriamay well contain a mixture of stimulatory and non-stimulatory LPSspecies, especially during transition between flea and hosttemperatures, with the antagonistic activity of tetra-acylated LPSblunting induction of innate immunity by the active LPS species.

Mass spectroscopy analysis showed mainly tetra-acylated Y. pestis lipidA in Y. pestis KIM5 grown at 37° C., with hexa-acylated structurespresent only at the lower temperature (FIG. 2A,B, FIG. 7A,B). LpxL(HtrB) and LpxM (MsbB) are “late” acyltransferases in E. coli andseveral other Gram-negative bacteria, adding the secondary acyl chainsto the tetra-acylated precursor lipid IV_(A) (6). LpxP, a third late E.coli acyltransferase, is active at low temperatures (6, 17, 18). Wecould identify only two late acyltransferases, lpxP and lpxM, in the Y.pestis genome (19-21). This suggests that the lack of LpxL together withthe temperature sensitivity of LpxP is responsible for the absence ofhexa-acylated lipid A at 37° C.

We cloned lpxL from E. coli K12, and expressed the gene in Y, pestisKIM5 under control of its own promoter on the vector pBR322 [Y. pestisKIM5(pMW::lpxL)](16). The nucleotide sequence of the promoter and thesequence encoding LpxL is set forth below (the promoter sequence and 3′untranslated region are set forth in lower case and the sequenceencoding LpxL is set forth in upper case):

(SEQ ID NO: 1) gcgttaatgccctcatgcgccagatacactcgcccaaaaacattcagcgcggtaaacagctgatataaagcgtcacgggtcgccttaggatcggcgatgtggaaatacttgtaaaacgaaatggtggttcgcggttcgctctcagccaacattttggcttttagcgcgtcgttggaaatgcggttgtgtaacactggcatggtgtacggttcctgcgagatgggaaagtaaaaatccgcggcatgatatagcaattatcgataattaacatccacacattttacgctacatttgcgcattaaaaattatttgttatttacaagcgcggcaatttcgcccagtcttcagccacaaattttggttgcgggcgaaaaaatgcgacaatacatacaattgcccgaataggttgaaaaacaggattgatATGACGAATCTACCCAAGTTCTCCACCGCACTGCTTCATCCGCGTTATTGGTTAACCTGGTTGGGTATTGGCGTACTTTGGTTAGTCGTGCAATTGCCCTACCCGGTTATCTACCGCCTCGGTTGTGGATTAGGAAAACTGGCGTTACGTTTTATGAAACGACGCGCAAAAATTGTGCATCGCAACCTGGAACTGTGCTTCCCGGAAATGAGCGAACAAGAACGCCGTAAAATGGTGGTGAAGAATTTCGAATCCGTTGGCATGGGCCTGATGGAAACCGGCATGGCGTGGTTCTGGCCGGACCGCCGAATCGCCCGCTGGACGGAAGTGATCGGCATGGAACACATTCGTGACGTGCAGGCGCAAAAACGCGGCATCCTGTTAGTTGGCATCCATTTTCTGACACTGGAGCTGGGTGCGCGGCAGTTTGGTATGCAGGAACCGGGTATTGGCGTTTATCGCCCGAACGATAATCCACTGATTGACTGGCTACAAACCTGGGGCCGTTTGCGCTCAAATAAATCGATGCTCGACCGCAAAGATTTAAAAGGCATGATTAAAGCCCTGAAAAAAGGCGAAGTGGTCTGGTACGCACCGGATCATGATTACGGCCCGCGCTCAAGCGTTTTCGTCCCGTTGTTTGCCGTTGAGCAGGCTGCGACCACGACCGGAACCTGGATGCTGGCACGGATGTCCGGCGCATGTCTGGTGCCCTTCGTTCCACGCCGTAAGCCAGATGGCAAAGGGTATCA ATTGATTATGCTGCCGCCAGAGTGTTCTCCGCCACTGGATGATGCCGAAACTACCGCCGCGTGGATGAACAAAGTGGTCGAAAAATGCATCATGATGGCACCAGAGCAGTATATGTGGTTACACCGTCGCTTTAAAACACGCCCGGAAGGCGTTCCTTCACGCTATTAAatctcccatgccggatgcttcagaatggcatccggcattaccacagcaaatccccctgatttagcgataaaagctctaggattgcgccccctggaagtcgggcgcataattagtgtgcttat.

The promoter sequence (sequence in lower case located 5′ to the sequenceencoding LpxL) is provided as SEQ ID NO: 2 and the nucleotide sequenceencoding the LpxL protein is provided as SEQ ID NO: 3.

The amino acid sequence of LpxL encoded by SEQ ID NOs: 1 and 3 is setforth below:

(SEQ ID NO: 4) MTNLPKFSTALLHPRYWLTWLGIGVLWLVVQLPYPVIYRLGCGLGKLALRFMKRRAKIVHRNLELCFPEMSEQERRKMVVKNFESVGMGLMETGMAWFWPDRRIARWTEVIGMEHIRDVQAQKRGILLVGIHFLTLELGARQFGMQEPGIGVYRPNDNPLIDWLQTWGRLRSNKSMLDRKDLKGMIKALKKGEVVWYAPDHDYGPRSSVFVPLFAVEQAATTTGTWMLARMSGACLVPFVPRRKPDGKGYQLIMLPPECSPPLDDAETTAAWMNKVVEKCIMMAPEQYMWLHRR FKTRPEGVPSRY.

Mass spectroscopy (FIG. 7C,D) shows that this strain containshexa-acylated structures at both 37° C. and 26° C., indicating that, asin E. coli, LpxL mediates addition of one 2′ secondary C12:0 acyl chainto lipid IV_(A). Presumably, a 3′ secondary C12:0 acyl moiety is addedby the endogenous LpxM. In contrast to results obtained with KIM5 (FIG.1, FIG. 3A), both 37° C. and 26° C. LPS from KIM5(pMW::lpxL) stronglyactivated HEK293 huTLR4/MD2 cells (FIG. 3A), but not TLR2-expressingcells (FIG. 3B). A similar observation was made with human PBMC: onlythe KIM5 37° C. LPS showed profoundly reduced induction of TNF, IL-6,and IL-8 release (FIG. 3C,D). Responses of human PBMC from severalhealthy volunteers (n=9) followed a similar pattern (not shown), as didPBMC from cynomolgus macaques (FIG. 8), indicating that primates havevery low ability to respond to native 37° C. Y. pestis LPS. Mousemacrophages released limited amounts of TNF, in a TLR4-dependent mannerwhen exposed to the 37° C. LPS from KIM5, but this response was muchweaker than observed with 37° C. LPS from KIM5(pMW::lpxL) or 26° C. LPSfrom either strain (FIG. 3E,F). Thus, a major deficit in response to 37°C. Y. pestis LPS was common to cells from all species tested.

Mammals have multiple survellience pathways that can triggerantibacterial responses. On the other hand, Y. pestis has sophisticatedactive mechanisms for suppressing these responses, including a type IIIsecretion system that intoxicates host cells in contact with thebacteria (1, 2). To determine the significance of LPS stimulatoryactivity in this complex environment, we conducted infection experimentsin mice. Virulent Y. pestis [strain KIM1001 (22)] induced 100% mortalityin mice after subcutaneous (s.c.) injection of 1000 c.f.u. (FIG. 4A). Incontrast, KIM1001(pMW::lpxL) caused neither mortality nor visibledistress (FIG. 4A). The effect of LpxL is profound: at doses of up to10⁷ c.f.u., approximately 10⁶ mean lethal doses for the KIM1001 parentstrain, no mortality or apparent signs of illness were detected (FIG.4B). Control experiments indicated that other Y. pestis virulencedeterminants were not affected by pMW::lpxL (Supporting text), and thatpBR322 by itself had no effect on virulence (FIG. 9). Although the mouseTLR4/MD-2 system has a limited but significant response totetra-acylated LPS/lipid A (FIG. 3E) (4, 23-25), it is apparentlyinsufficient to protect infected mice. Our data suggest that thedevelopment of bubonic plague is dependent on the production of LPS withreduced TLR4/MD-2 stimulating activity.

This hypothesis predicts that resistance to KIM1001(pMW::lpxL) should beTLR4-dependent. Accordingly, we infected both wild type and TLR4−/− micewith KIM1001 or KIM1001(pMW::lpxL) with a s.c. dose of 1000 c.f.u. Wildtype and TLR4−/− animals succumbed to infection with KIM1001, while onlyTLR4−/− animals died when infected with KIM1001(pMW::lpxL) (FIG. 4C). Incontrast, TLR4−/− animals survived infection with a Y. pestis Plamutant, a strain that does not cause systemic infection by s.c. route(22), indicating that the TLR4−/− animals do not harbor a general defectin defense against other avirulent Y. pestis strains (Supporting text).

Foci of infection containing virulent Y. pestis are often devoid ofinflammatory cells, reflecting the ability of this pathogen to evadeimmune responses. Intravenous (i.v.) infection is a useful model forexamining inflammation in tissues remote from injection site trauma.When mice are infected i.v. with KIM1001(pMW::lpxL), enhancedanti-bacterial response is revealed by extended survival time (FIG. 10)and a 100-fold reduction (p=0.008) in spleen bacterial titers (FIG. 4D).Despite lower bacterial load, KIM1001(pMW::lpxL) infected animals hadtwo-fold more TNF in spleen homogenates compared to mice infected withKIM1001 (FIG. 4E). Livers of mice infected with wild type Y. pestis arecharacterized by masses of free bacteria, without distinct indicationsof local inflammation (FIG. 4F). In contrast, KIM1001(pMW::lpxL)provokes microabcess formation in livers from wild type mice, but not inTLR4−/− animals (FIG. 4F). Liver tissue was normal in animals receiving1000 c.f.u. of KIM1001(pMW::lpxL) s.c., (FIG. 4F), and no bacteria weredetected in spleens (FIG. 4E). This indicates that s c administration ofKIM1001(pMW::lpxL) fails to establish systemic infection, presumably dueto containment by effective local immunity.

We hypothesized that the avirulent Y. pestis (pMW::lpxL) producingpotent LPS might be effective as a vaccine against plague. Mice werevaccinated with a single s.c. dose (1000 c.f.u.) of KIM1001(pMW::lpxL).Thirty-five days later, both vaccinated and naïve mice were challengedwith virulent KIM1001 at an s.c. dose of 1000 c.f.u. All mice vaccinatedwith KIM1001(pMW::lpxL) survived while all naïve mice died (FIG. 4G),demonstrating that Y. pestis producing potent LPS is an effectivevaccine.

In another experiment, mice were vaccinated s.c. with 100000 c.f.u. ofKIM1001(pMW:::lpxL), and re-challenged with 100000 and 1000000 c.f.u. ofKIM1001, days later. 5 out of 5 naïve mice die when infected withKIM1001, 100000 c.f.u., but 0 out of 5 mice died when they arevaccinated as given and re-challenged 30 days later with either 100000or 1000000 clu. of virulent KIM1001. These results indicate that themodified Y. pestis strain may be used as a vaccine against bubonicplague.

The following experiment shows that modified Y. pestis may also be usedas a vaccine against pneumonic plague. Mice (wild type C57B1/6) werevaccinated s.c. with 100000 c.f.u. of Y. pestis KIM1001(pMW:::lpxL) inthe nape of the neck. 30 days later, the mice were challengedintranasally (i.n.) with 5000 or 50000 c.f.u. of virulent Y. pestisKIM1001. Intranasal challenge is a pneumonic plague model. Survival wasmonitored (five mice per vaccinated group, 8 mice in unvaccinated). Theresults, which are shown in FIG. 13, indicate that Y. pestisKIM1001(pMW:::lpxL) effectively protected mice against pneumonic plague.

Thus, these results indicate that generation of avirulent bacterialstrains harboring enhanced TLR-activating potential could be a usefulstrategy for vaccine production.

The general picture that emerges from our current understanding of Y.pestis virulence is that successful systemic infection is dependent onefficient and simultaneous suppression and evasion of multiple pathwaysordinarily capable of eliciting protective local inflammatory responses(1, 2, 22). Our results establish that evading activation of TLR4signaling is an essential part of the Y. pestis strategy. These resultsalso emphasize the remarkable efficiency of TLR4-mediated responses inthe control of Gram-negative infections. Several other Gram-negativepathogens have been shown to synthesize lipid A with weak stimulatoryactivity, associated with differences in number and length of acylchains (9, 10). To our knowledge, this is the first report to establishthe significance of the synthesis of such lipid A for pathogenesis.

The strong TLR4-mediated protection against Y. pestis producing potentLPS suggests that the development of anti-infectives based onstimulation of innate immunity, now in its infancy, has significantpromise (26, 27). However, incorporation of adjuvant activity intopathogens by LPS modification or other means may also be effectivelyapplied as a novel principle for vaccines (28).

REFERENCES

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Materials and Methods: Bacterial Strains and Growth Conditions:

The lpxL gene from E. coli K12, including 435 by upstream and 140 bydowstream of coding region, was cloned using Pfu Ultra polymerase(Stratagene) and ligated into the BamHI and SalI sites of pBR322,creating pMW::lpxL. The control plasmid pBR322Δtet was constructed bydigesting empty pBR322 with Nae1 and EcoRV and then ligating the plasmidto remove the major part of the tetracycline resistant gene. Theresulting plasmids were electroporated into Yersinia pestis strain KIM5(Goguen et al., (1984) J Bacteriol 160, 842-8), or KIM1001 (Sodeinde etal., (1992) Science 258, 1004-7) and selected by growth on tryptose-beefextract (TB) agar supplemented with 2.5 mM CaCl₂ and 0.6 mg/mL glucosein the presence of 100 μg/ml ampicillin. All strains containing eitherpBR322Δtet or pMW::lpxL remained tetracycline sensitive. KIM1001(pPCP1+, pCD1+, pMT1+) is highly virulent (Sodeinde et al., (1992)Science 258, 1004-7), while the KIM derivative KIM5 bears a chromosomaldeletion designated Δpgm, which strongly attenuates virulence. KIM5 wasused to limit risk of infection in in vitro studies where a virulentstrain was not required, and the pgm locus contains no genes thought toimpact LPS biosynthesis. For LPS or lipid A preparations, KIM5 andKIM5(pMW::lpxL) were grown overnight at the indicated temperatures inaerated TB broth supplemented with CaCl₂ and glucose as given above. Forthe generation of heat killed bacteria, cultures were resuspended in PBSand incubated at 60° C. for 1 hr.

Lipid Preparation:

Pyrogen-free reagents and supplies were used to the greatest extentpossible in the lipid preparations. Bacteria were harvested bycentrifugation at 6000×g. Lipid A was directly isolated from wholebacterial cells using the Bligh-Dyer two-phase chloroform-methanol-waterorganic extraction (Vorachek-Warren et al., (2002) J Biol Chem 277,14186-93). Samples were subsequently analyzed by MALDI-TOF MS using aKratos (Manchester, UK) AXIMA CFR high performance mass spectrometeroperated in both positive and negative ion modes. LPS was purified frombacteria by hot water-phenol extraction (Westphal et al., (1952)Naturforsch. B7, 148-155), followed by two phenol re-extractions toremove contaminating lipoproteins and TLR2-activity (Hirschfeld et al.,(2000) J Immunol 165, 618-622). The chemical synthesis of thetetra-acylated precursor lipid IVA (also called 406, LA-14-PP orprecursor Ia) has been reported (Liu et al., (1999) Bull. Chem. Soc.Jpn. 72, 1377-1385). LPS from E. coli O111:B4 (Sigma) was phenolre-extracted as indicated above. The LPS preparations were unable tostimulate HEK293 huTLR2-expressing cells, in spite of strong cellularactivation by Pam3CysSK4 lipopeptide, indicating the absence ofcontaminating lipoproteins in our LPS (FIG. 3B and not shown).

Cell Stimulation Assays:

Human PBMC were obtained from healthy volunteers and isolated bycentrifugation on Lympoprep density media (Axis-Shield/Nycomed, Oslo,Norway). Cynomolgous macaque PBMC, purified by centrifugation of wholeblood in CPT/sodium citrate tubes (Becton Dickinson), was obtained fromBioreclamation, Inc (Hicksville, N.Y.). HEK293 cells stably expressinghuman TLR4-YFP and retroviral MD-2, or human TLR2-YFP, or empty vectorpcDNA3 were as published (Latz et al., (2002) J Biol Chem 277,47834-43). No activation of the HEK293 huTLR2-expressing cell line wasobserved by our LPS preparations, in spite of strong activation byPam3CysSK4 lipopeptide, suggesting absence of contaminating lipoproteinin the LPS. NF-κB-luciferase (provided by K. Fitzgerald) andIRF-3-dependent 561-luciferase [a gift from G. Sen (Bandyopadhyay etal., (1995) J Biol Chem 270, 19624-9)] reporters were transfected into293-huTLR4/MD-2 cells using Genejuice (Novagen). PBMC were cultured inX-vivo 15 medium (Cambrex) containing ciprofloxacin and 1% FCS or 1%human serum, whereas 293 cells were stimulated in DMEM/ciprofloxacinplus 10% FCS. Wild type C57B1/6 mice were from Jackson Laboratories (BarHarbor, Me.). TLR4−/− animals, a gift from Dr. S. Akira, were generatedas described (Hoshino et al., (1999) J Immunol 162, 3749-52), andbackbred 11 generations into C57B1/6. Mouse peritoneal macrophages wereharvested from wild type C57B1/6 or TLR4−/− mice 3 days after injectionof 2 ml thioglycollate (3%) and cultured in RPMI 1640 medium containing10% FCS. Cells were plated at a density of 2×10⁴ cells/well (293 cells)5×10⁴ cells/well (mouse macrophages) or 1×10⁵ cells/well (PBMC) in96-well dishes, and stimulated 16-18 hrs before harvesting supernatant(for cytokine analysis) or cells for lysis (transfections and reporterassays, using reagents from Promega). Cytokines were measured usingELISA kits from BD Pharmingen (moTNFα) or R&D systems (huTNFα, IL-6,IL-8). Cell lysates from luciferase reporter assays were analyzed by theaddition of luciferase substrate followed by luminometry. Results areshown as one representative out of three to eight experiments, as meanof triplicates (transfection assays) or triplicates/duplicates (cytokineassays)+/−standard deviation (s.d.).

In Vivo Infections:

Mice were infected with Y. pestis KIM1001, KIM1001(pMW::lpxL) orKIM1001(pBR322Δtet), either by s.c. injection of 50 μl on the nape ofthe neck or i.v. injection of 500 μl in the tail vein. Inocula containedthe indicated c.f.u. suspended in PBS. Survival was monitored up to 21days, every 12 hrs during acute infection. For collection of organs,mice were sacrificed 48 hrs following i.v. infection or 72 hrs followings.c. infection by pentobarbital overdose, spleens were homogenized inPBS for bacterial titers. Separate infection experiments with detectionlimits of 10³ or 10¹ c.f.u. per spleen showed similar results, with theabsence of detectable bacteria in spleens following s.c. infection withKIM 1001 (pMW::lpxL). Livers were fixed for 3 days in neutral buffered4% formalin, followed by standard hematoxylin/eosin (H&E) staining andmicroscopy (100× magnification). Inserts [KIM1001 and KIM1001(pMW::lpxL)i.v.] are at 1000× magnification. All animal studies were approved bythe Institutional Animal Care and Use Committee. Differences in spleenc.f.u. and TNF concentrations were analyzed by Mann-Whitney U-test.Statistical differences in survival were studied by Kaplan-Meyersurvival analysis and the log rank test.

Supporting Text:

Y. pestis Grown at 37° Poorly Activates TLR4 Signaling

When grown at 26° C., heat-killed Y. pestis KIM5, an attenuated straindeficient in iron acquisition, strongly activates HEK293 huTLR4/MD-2cells to release IL-8 and to activate transcription factors NF-κB andIRF-3 (FIG. 9 a, b). If the bacteria are grown at 37° C., thisactivation is not observed. Activation and nuclear translocation ofthese two transcription factors constitute two major branches of TLR4signaling necessary for the induction of pro-inflammatory cytokines andtype I interferons (Akira et al., (2004) Nat Rev Immunol 4, 499-511;Liew et al., (2005) Nat Rev Immunol 5, 446-58). These data, togetherwith the presence of poorly immune activating tetra-acylated Y. pestislipid A structures at 37° C. (Kawahara et al., (2002) Infect Immun 70,4092-8; Rebeil et al., (2004) Mol Microbiol 52, 1363-73), suggest thatevasion of TLR4-signaling could represent a central mechanism by whichY. pestis avoids innate immune activation.

Y. pestis Containing (pMW::lpxL) Retains Key Features

To further ensure that important virulence-related functions of Y.pestis that might be altered by changes in membrane structure were notaffected by lpxL, we confirmed that pMW::lpxL did not affect thefunction of type III secretion activity as evidenced by secretion ofYopM, retention of cytotoxic activity for HeLa cells in vitro, andretention of Ca⁺⁺-dependent growth at 37° C., and also did not affectthe ability of the outer membrane protease Pla to act on the highmolecular weight substrate plasminogen. We also determined thatpMW::lpxL was stable in the absence of selection (no plasmids segregantsdetected among 1000 colonies after 40 generations of growth in liquidculture), and that native Y. pestis plasmids were retained in thepresence of pMW::lpxL.

Bacteria obtained from spleens of mice infected i.v. withKIM1001(pMW:lpxL) all remained ampicillin resistant following patchingof 100 colonies. Furthermore, the presence of the control plasmidpBR322Δtet in KIM1001 did not affect virulence in mice (FIG. 9).

TLR4−/− Mice Survive Infection with Pla Mutant Y. pestis

Y. pestis KIM1001(pMW::IpxL) is avirulent in wt mice, but fully virulentin TLR4−/− animals. We also considered the possibility that TLR4deficiency would alter the susceptibility to other types of Y. pestisstrains that are avirulent by s.c. infection. To test this, we infectedmice with Y. pestis KIM lacking the outer membrane protease known asplasminogen activator (Pla) (Sodeinde et al., (1992) Science 258,1004-7). Pla clearly contributes to suppressing local inflammation,although the mechanism of this contribution is currently not known. Wehave previously shown that this strain, like KIM expressing lpxL, isavirulent by s.c. infection but virulent when given i.v., and provokessubstantial local inflammation (Sodeinde et al., (1992) Science 258,1004-7). Thus, survival of infection with this strain also depends onvigorous local innate responses. If the effect of TLR4 on innateresponses is non-specific, TLR4-deficient mice should be susceptible toPla⁻ Y. pestis. All of three TLR4−/− mice infected with the Pla⁻ strain(Sodeinde et al., (1992) Science 258, 1004-7) s.c. at 10⁵ c.f.u.survived, showed no signs of illness, and were immune to later challengewith wild type KIM confirming infection. This result indicates that TLR4deficiency does not grossly compromise innate responses to Y. pestis ingeneral, and that loss of Pla and production of stimulatory LPS, whileboth permitting effective innate responses, do so via distinct pathways.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1-13. (canceled)
 14. A method for protecting a mammal from infection bya gram-negative pathogen, comprising administering to a mammal aneffective amount of a vaccine that comprises at least about 100 colonyforming units (c.f.u.) of a modified gram-negative bacterium thatcomprises low potency lipopolysaccharide (LPS) comprising one or moreheterologous nucleic acids encoding one or more enzymes that permits thebacterium to produce high potency LPS.
 15. A method for vaccination of amammal against the plague, comprising administering to the mammal atherapeutically effective amount of a modified Yersinia pestis bacteriumthat comprises low potency lipopolysaccharide (LPS) comprising one ormore heterologous nucleic acids encoding LPS biosynthetic enzyme LpxLthat permits the bacterium to produce hexa-acylated high potency LPS.16-19. (canceled)
 20. A method for delivering an antigen to a subject,comprising delivering to a subject a therapeutically effective amount ofa microorganism modified to express an immunostimulatory molecule,wherein the microorganism further comprises an antigen.
 21. A method forprotecting a mammal from infection by a gram-negative pathogen,comprising administering to a mammal an effective amount of a vaccinecomprising at least about 100 colony forming units (c.f.u.) of atransgenic gram-negative bacterium that is derived from a wild-typebacterium that does not comprise high potency lipopolysaccharide (LPS),wherein said transgenic bacterium comprises said high potencylipopolysaccharide (LPS) and comprises a heterologous nucleic acidsequence encoding an acyltransferase that permits the transgenicbacterium to produce high potency LPS, a) wherein the heterologousnucleic acid sequence is at least about 80% identical to a nucleic acidsequence comprising SEQ ID NO: 1, b) wherein said nucleic acid sequenceencodes a LPS biosynthetic enzyme LpxL, c) wherein said LpxL comprisesan amino acid sequence which is at least about 90% identical to SEQ IDNO: 4, and d) wherein said transgenic bacterium has reduced virulencerelative to a control bacterium that contains a mutation in saidheterologous nucleic acid sequence.
 22. The method of claim 21, whereinthe high potency LPS activates Toll-like receptor 4 (TLR4) in mammaliancells.
 23. The method of claim 22, wherein the high potency LPS ishexa-acylated LPS.
 24. The method of claim 23, wherein the bacterium isselected from the group consisting of Yersinia pestis, Chlamydiatrachomatis, Francisella tularensis, Legionella pneumophila, Brucellaabortus and Chlamydia pneumoniae.
 25. The method of claim 24, whereinsaid transgenic bacterium is Yersinia pestis.
 26. The method of claim25, wherein the enzyme LpxL is from E. coli and comprises SEQ ID NO: 4.27. The method of claim 21, wherein said transgenic bacterium is alive.28. The method of claim 21, wherein said transgenic bacterium is killed.29. The method of claim 21, wherein said heterologous nucleic acidsequence comprises a nucleotide sequence which is at least about 85%,90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence comprising the nucleotide sequence of SEQ ID NO:
 1. 30. Themethod of claim 21, wherein said transgenic bacterium has at least10-fold reduced virulence relative to said control bacterium.
 31. Themethod of claim 30, wherein said transgenic bacterium is Yersinia pestisand is avirulent in a wild type mouse.
 32. The method of claim 21,wherein said heterologous nucleic acid sequence is SEQ ID NO: 1, andsaid LpxL is SEQ ID NO:
 4. 33. A method for protecting a mammal frominfection by a gram-negative pathogen, comprising administering to amammal an effective amount of a vaccine comprising at least about 100colony forming units (c.f.u.) of a transgenic gram-negative bacteriumthat is derived from a wild-type bacterium that does not comprise highpotency lipopolysaccharide (LPS), wherein said transgenic bacteriumcomprises a heterologous nucleic acid sequence encoding anacyltransferase, a) wherein the heterologous nucleic acid sequence is atleast about 80% identical to a nucleic acid sequence comprising SEQ IDNO: 1, b) wherein said nucleic acid sequence encodes a LPS biosyntheticenzyme LpxL, c) wherein said LPS biosynthetic enzyme LpxL comprises anamino acid sequence which is at least about 90%, 92%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence comprising theamino acid sequence of SEQ ID NO: 4, and d) wherein said transgenicbacterium has reduced virulence relative to a control bacterium thatcontains a mutation in said heterologous nucleic acid sequence.
 34. Amethod for protecting a mammal from infection by a gram-negativepathogen, comprising administering to a mammal an effective amount of avaccine comprising at least about 100 colony forming units (c.f.u.) of atransgenic gram-negative bacterium that is derived from a wild-typebacterium that does not comprise high potency lipopolysaccharide (LPS),wherein said transgenic bacterium comprises said high potencylipopolysaccharide (LPS), and comprises a heterologous nucleic acidsequence encoding an acyltransferase that permits the transgenicbacterium to produce high potency LPS, a) wherein said heterologousnucleic acid sequence is at least about 80% identical to a nucleic acidsequence comprising SEQ ID NO: 1, b) wherein said nucleic acid sequenceencodes a LPS biosynthetic enzyme LpxL, c) wherein said LpxL comprisesan amino acid sequence which is at least about 90% identical to SEQ IDNO: 4, d) wherein said transgenic bacterium has reduced virulencerelative to a control bacterium that contains a mutation in saidheterologous nucleic acid sequence, and e) wherein said wild-typebacterium is selected from the group consisting of Yersinia pestis,Chlamydia trachomatis, Francisella tularensis, Legionella pneumophila,Brucella abortus and Chlamydia pneumoniae.